Background
==========

Osteoarthritis (OA) is the most common joint disease. Articular cartilage in OA has shown to lose its mechanical resistance, elasticity and smoothness, and is consequently worn out by the movements of the joint. This leads to reactive bone remodeling, forming osteophytes, microfractures, subchondral eburnation and pseudocysts, and exposure of the articular end of the bone \[[@B1]\]. The consequent roughness of the articular cartilage surfaces elicits secondary inflammatory reactions of the synovial membrane and bone. Unlike rheumatoid arthritis and other inflammatory joint diseases, the inflammatory component of OA is relatively mild \[[@B2],[@B3]\]. Clinical manifestations of OA of the knee are joint pain, stiffness in the morning or after rest, pain at night, limited joint motion and/or joint deformity. Joint pain in OA may originate from not only synovitis, but also stretching of the joint capsule or ligaments, periosteal irritation due to osteophyte formation, trabecular microfractures, intraosseous hypertension, or muscle spasm \[[@B4]-[@B7]\].

There are many treatment modalities for OA of the knee including nonpharmacologic (e.g. patient education, weight control, physical and occupational therapy, and aerobic exercise programs) and pharmacologic therapy (e.g. intraarticular steroid injections, paracetamol, topical analgesics, nonsteroidal anti-inflammatory drugs (NSAIDs) and opioid analgesics) \[[@B8]\]. Although NSAIDs are the most widely prescribed drugs to reduce joint pain and stiffness, the inflammatory component of OA is usually minimal, therefore, the need for the anti-inflammatory effect of NSAIDs in this condition is controversial \[[@B2],[@B9],[@B10]\]. Moreover, inhibition of prostaglandin biosynthesis is directly related to many common and occasionally severe side effects including gastrointestinal bleeding, hypertension, congestive heart failure, hyperkalemia, and renal insufficiency \[[@B11]-[@B14]\]. These disadvantages call for an evaluation of the risks and benefits of the therapy in comparison with a less toxic one for OA.

Since the efficacy of NSAIDs in symptomatic treatment of OA of the knee depends on the analgesic rather than anti-inflammatory effect, paracetamol (analgesic drug) has been recently recommended as the first-line oral drug in the management of OA of the knee \[[@B8]\]. However, the long term use of paracetamol probably leads to hepatic and renal impairment \[[@B15],[@B16]\]. Thus, less toxic pain managing procedures, e.g. electroacupuncture (EA), may be considered as an alternative treatment of this disease. Even though EA is safe and effective in combating pain \[[@B17],[@B18]\], its role in OA of the knee is still controversial and comparative studies between EA and NSAIDs in this disease are rare \[[@B19]\]. Therefore, the aim of this study was to compare the efficacy of EA, diclofenac and their combination in short-term, symptomatic treatment of OA of the knee.

Materials and methods
=====================

Research design
---------------

This study was a randomized, single-blind, placebo controlled trial. The treatment procedures consisted of placebo tablet plus placebo EA (placebo group), diclofenac tablet plus placebo EA (diclofenac group), placebo tablet plus true EA (EA group) and diclofenac tablet plus true EA (combined group). This study was approved by the Medical Ethics Committee of the Faculty of Medicine, Chiang Mai University and was in compliance with the Helsinki Declaration.

Subjects
--------

Two hundred out-patients of either sex, aged over 40 years, and who had been suffering from unilateral or bilateral OA of the knee according to the criteria of the American College of Rheumatology \[[@B8]\] for more than 3 months duration, were recruited. Lequesne\'s functional index, which was evaluated at the screening visit, had to be at least 6 points. Subjects had to be able to walk and give both verbal and written information regarding the study. Signed informed consent was obtained prior to entry. Exclusion criteria included an underlying inflammatory arthropathy, expectation of surgery in the future, recent injury in the area affected by OA of the knee, intraarticular corticosteroid injections or EA within the last 3 months, hypersensitivity to NSAIDs or paracetamol, abnormal liver or kidney function tests, evidence of leukopenia and coagulopathies screened by clinical laboratory, concomitantly receiving anticoagulants, history of peptic ulceration, anemia, uncontrolled hypertension, congestive heart failure, hyperkalemia, pregnancy, lactation and malignant tumors.

Treatment procedures
--------------------

### 1. Drug administration

During a run-in period of one week, the patients refrained from using any NSAIDs or analgesics except for a \"rescue analgesic\" (2 tablets of 500 mg paracetamol orally as needed, up to 4 times daily). The patients who had persistent pain and a Lequesne\'s functional index of at least 6 points at the end of the run-in period were randomized into the four groups mentioned above. Diclofenac sodium, 25 mg film-coated tablets were a gift from Novartis (Thailand) Limited. Placebo and diclofenac were prepared in identical appearance. Either the placebo or diclofenac was prescribed at 1 tablet, 3 times a day immediately after each meal for 4 weeks. In addition, 2 tablets of 500 mg paracetamol were still prescribed as a \"rescue analgesic\" during this study.

### 2. True and placebo EA

The true EA treatment was standardized throughout the study. It was performed by the physician acupuncturist who received acupuncture training in the People\'s Republic of China. Four fine stainless steel needles were inserted into acupuncture points around the affected knee \[[@B20]\] as presented in Table [1](#T1){ref-type="table"} and Figure [1](#F1){ref-type="fig"}. All needles were used in order to conduct an electrical current through the points, and were inserted superficially (not more than 0.5 inch approximately in depth). Thus, an elicitation of needle sensation (so-called De Qi) during the insertion of the needles was not intended. The first pair of electrodes was connected to the Dubi and nearest adjacent point (medial Xiyan) and the second pair to the trigger point and Ququan. The electrical stimulation was applied slowly and simultaneously to each pair of needles until it reached the maximum toleration level of the patient. Biphasic pulses were used for the electrical stimulation at a frequency of 2 Hz, and it was administered for 20 minutes in each treatment. The patients were treated 3 times a week (Monday, Wednesday, and Friday) for 4 weeks (12 times).

![The selected acupuncture points around the knee, demonstrated in sitting position, during treatment with true (A) and placebo EA (B).](1472-6882-2-3-1){#F1}

::: {#T1 .table-wrap}
::: {.caption}
######

The selected acupuncture points used in this study.
:::

  Acupuncture points        Location                                                                                Needling manipulation
  ------------------------- ----------------------------------------------------------------------------------------------------------------- ------------------------------------------
  Dubi (ST-35)^1^           In the depression of the lateral part of the patella ligament, when the knee is bent.                             Slightly towards the medial side^\*^.
  Medial Xiyan (Extra)^1^   In the depression of the medial part of the patella ligament, when the knee is bent.                              Slightly towards the lateral side^\*\*^.
  Trigger point^2^          At the level of the joint line, midpoint between the medial Xiyan and Ququan.                                     Straight insertion^\*^.
  Ququan (Liv-8)^2^         At the medial end of the knee crease, in front of the semi-membranous muscle behind the lower end of the femur.   Straight insertion^\*\*^.

^1^First pair of elctrodes ^2^Second pair of electrodes ^\*^Stimulated with positive polarity at visit \#1, 3, 5, 7,9,11 and negative polarity at visit \#2, 4, 6, 8, 10, 12. ^\*\*^Stimulated with negative polarity at visit \#1, 3, 5, 7,9,11 and positive polarity at visit \#2, 4, 6, 8, 10, 12.
:::

The placebo EA was performed by attaching patch electrodes to the selected acupuncture points. Each electrode was connected to the sound producing dummy mode of the same apparatus, as in the true EA treatment. The duration and frequency of treatment were the same as those in the true EA treatment. Both true and placebo EA were performed by the same physician. Thus, the physician acupuncturist was the only person in the research team who knew which patients received the true or placebo EA.

Compliance with treatment was assessed by counting the number of unused tablets (diclofenac or placebo) and the number of times acupuncture treatment was received. During the study period, all additional therapies (e.g. oral or topical NSAIDs, intraarticular corticosteroid injection, other analgesics, chondro-protective agents, surgical procedures on the knee joint etc.) were not allowed. However, all other treatment for concomitant disorders that did not interfere with the study could be continued, but it had to be documented.

### Assessments

Clinical assessments were evaluated for base-line data at the end of the run-in period (week 0) and again at the end of the study (week 4). These assessments included the amount of paracetamol tablets taken/week, 50 feet-walk time, a patient\'s global pain as 100 mm visual analog scale (VAS) over the previous 3 days, the Western Ontario and McMaster Universities OA Index (WOMAC: score ranging from 0--96) \[[@B21]\], and Lequesne\'s functional index (score ranging from 0--24) \[[@B22]\]. At the end of this study, the orthopedist\'s and patient\'s opinion of change (much better, better, same, worse, much worse) were evaluated. The patients were considered to be responders if they met the following criteria. Firstly, the number of paracetamol tablets taken in week 4 was less than that at week 0, or less than 14 tablets/week. Secondly, at least 4 of the following 5 outcome parameters showed the following improvement: VAS, WOMAC, Lequesne\'s functional index decreased by at least 50%, and the orthopedist\'s or patient\'s overall opinion of change was better or much better. Clinical assessments in each patient were evaluated by the same physician who was blinded to the treatment. Complete physical examination and non-directive questioning for adverse events were also performed weekly for 4 weeks in order to acquire a safety assessment.

Statistical analysis
--------------------

One-way analysis of variance (ANOVA) was used to determine whether the four treatment groups differed in mean values of change from a base-line in paracetamol consumption, 50 feet-walk time, VAS, WOMAC and Lequesne\'s functional index. If there was any statistical significance between any of the four groups, the Scheffe method was used to demonstrate statistical significance between each of two groups. Differences among the treatment groups in the overall opinion of change, and number of patients considered to be responders, were evaluated by chi-square or Fisher\'s exact test.

Results
=======

Of the 200 subjects considered eligible for the study, 5 dropped out and 2 were withdrawn during the run-in period due to hypersensitivity to paracetamol and spontaneous pain relief. The remaining 193 subjects constituted the study population and were randomized into four parallel groups, which were not significantly different in base-line characteristics (Table [2](#T2){ref-type="table"}) and radiographic findings of affected knees (Table [3](#T3){ref-type="table"}). Only 1 patient in each placebo, EA and combined group as well as 2 patients in the diclofenac group received acupuncture treatment prior to this study.

::: {#T2 .table-wrap}
::: {.caption}
######

Base-line characteristics of patients evaluated at the end of the run-in period.
:::

                                                     Treatment groups
  -------------------------------------------------- ------------------ ---------------- --------------- --------------- ---------
  n (M:F)                                            47 (12:35)         49 (11:38)       48 (10:38)      49 (10:39)      0.93^c^
  Age (yr)^\*^                                       62.70 (7.22)       62.14 (7.53)     65.10 (3.40)    61.84 (8.95)    0.16^a^
  Body weight (kg)^\*^                               60.65 (10.24)      57.65 (10.64)    59.89 (9.74)    59.92 (9.66)    0.49^a^
  Height (cm)^\*^                                    153.94 (6.45)      151.94 (10.71)   152.19 (5.89)   153.32 (6.73)   0.54^a^
  Duration of OA (yr)^\*^                            4.98 (3.32)        3.94 (2.83)      6.09 (4.96)     4.53 (3.86)     0.05^a^
  Localization of OA                                                                                0.40^c^
  Right/Left knee                                    2/3                2/6              1/4             3/5
  Both knees                                         42                 41               43              41
  Number of paracetamol tablets taken per week^\*^   22.06 (13.75)      18.94 (14.68)    21.40 (14.97)   19.04 (14.87)   0.63^a^
  50 ft-walk time (sec)^\*^                          22.04 (4.81)       22.36 (6.00)     24.54 (8.14)    22.77 (5.13)    0.21^a^
  VAS^\*^                                            63.49 (22.36)      64.79 (23.41)    66.87 (22.34)   57.63 (21.21)   0.21^a^
  WOMAC^\*^
   Pain index                                        10.19 (4.20)       11.02 (4.15)     10.25 (3.86)    10.50 (4.18)    0.75^a^
   Stiffness index                                   4.51 (1.71)        4.08 (1.95)      4.35 (2.10)     4.27 (1.57)     0.72^a^
   Disability index                                  37.04 (12.00)      35.65 (12.89)    38.00 (13.18)   37.94 (13.02)   0.79^a^
   Total score                                       51.75 (15.96)      50.76 (17.98)    52.60 (18.13)   52.71 (17.65)   0.94^a^
  Lequesne\'s functional index^\*^                   13.78 (3.78)       13.85 (3.22)     14.14 (2.98)    13.73 (2.92)    0.93^a^

^\*^Data represent mean (SD) ^\*\*^Statistic analysis: ^a^ = one-way ANOVA, ^c^ = chi-square test.
:::

::: {#T3 .table-wrap}
::: {.caption}
######

The radiographic findings at entry into the study^\*^.
:::

                                                   Treatment groups
  ------------------------------------------------ ------------------ ---- ---- ---- ---------
  Kellgren and Lawrence X-ray grade \[[@B32]\]
   Grade 1                                         4                  1    3    5    0.40^f^
   Grade 2                                         4                  6    5    7    0.84^f^
   Grade 3                                         19                 15   25   23   0.31^c^
   Grade 4                                         62                 68   58   55   0.16^c^
  Knee compartment with most severe change of OA
   Medial tibiofemoral                             63                 77   69   64   0.73^c^
   Lateral tibiofemoral                            12                 5    13   11   0.24^c^
  Patellofemoral                                   14                 8    9    15   0.28^c^

Data represent number of patients. Statistic analysis: ^c^ = chi-square test, ^f^ = Fisher\'s exact test.
:::

Of the 193 study patients, 186 (96.37%) completed the study. The remaining 7 patients were withdrawn from the trial due to flare of pain with joint swelling (2 in the placebo and 1 in the EA group), severe GI side effects (3 in the combined group), and flare of pain from an accidental fall not related to treatment (1 in the EA group). Since there were few patients withdrawn from the trial, the results were, therefore, not substantially affected, whether the analysis was performed by an intention to treat analysis or an analysis on available completers. Thus, the following data showed the findings in only 186 available completers.

During 4 weeks of treatment, change in body weight compared to the base-line values did not significantly differ among the four groups (one-way ANOVA calculated weekly, data not shown). The rates of compliance with medications (placebo or diclofenac) and acupuncture (placebo or EA) in each group were more than 90%, and comparable among the four groups.

At the end of the study, the improvement of symptoms, which were determined by the reduction in mean changes in most outcome parameters (except WOMAC pain index and WOMAC total score), was greatest in the EA group (Table [4](#T4){ref-type="table"}). The mean changes in VAS were significantly different between the EA and placebo group as well as the EA and diclofenac group (Table [4](#T4){ref-type="table"} and Figure [2](#F2){ref-type="fig"}). The mean changes in the Lequesne\'s functional index also differed significantly between the EA and placebo group (Table [4](#T4){ref-type="table"}, Figure [3](#F3){ref-type="fig"}). In addition, there were significant differences in mean changes in the subscale of standardized WOMAC (pain index) between the combined and placebo group (Table [4](#T4){ref-type="table"} and Figure [4](#F4){ref-type="fig"}). The proportion of patients with the orthopedist\'s and patient\'s overall opinion of \"much better\" was greatest in the EA group, followed by the diclofenac, combined and placebo group, respectively. In addition, the proportion of responders was also greatest in the EA group, followed by the combined, diclofenac and placebo group, respectively. However, statistical differences among the four groups were found only in the orthopedist\'s overall opinion and a number of responders (Table [5](#T5){ref-type="table"}). There were no differences between the four groups in the remaining outcome parameters.

![Change in 100 mm VAS in each treatment group evaluated at the end of week 4. Values are mean ± SEM. ^\*^ = *p* \< 0.05 between groups (one-way ANOVA and Scheffe method).](1472-6882-2-3-2){#F2}

![Change in Lequesne\'s functional index in each treatment group evaluated at the end of week 4. Values are mean ± SEM. ^\*^ = *p* \< 0.05 between groups (one-way ANOVA and Scheffe method).](1472-6882-2-3-3){#F3}

![Change in WOMAC pain index in each treatment group evaluated at the end of week 4. Values are mean ± SEM. ^\*^ = *p* \< 0.05 between groups (one-way ANOVA and Scheffe method).](1472-6882-2-3-4){#F4}

::: {#T4 .table-wrap}
::: {.caption}
######

Change in outcome parameters after 4 weeks of treatment^\*^.
:::

                                                         Treatment groups
  ------------------------------------------------------ ------------------ --------------- --------------- --------------- -------------
  Number of paracetamol taken (tablets taken per week)   -5.16 (2.33)       -4.43 (1.90)    -7.89 (2.09)    -5.13 (2.06)    NS
  50 feet-walk time (sec)                                -2.70 (0.52)       -3.52 (0.46)    -4.41 (0.70)    -4.13 (0.54)    NS
  VAS                                                    -22.86 (4.02)      -32.99 (3.94)   -48.24 (3.59)   -35.59 (2.74)   \<0.05^†^
  WOMAC
   Pain index                                            -3.31 (0.68)       -4.90 (0.53)    -5.65 (0.59)    -6.28 (0.77)    \<0.05^††^
   Stiffness index                                       -1.47 (0.31)       -1.55 (0.27)    -2.24 (0.31)    -2.02 (0.28)    NS
   Disability index                                      -12.33 (1.88)      -14.39 (1.77)   -19.17 (2.05)   -18.98 (1.92)   NS
   Total score                                           -17.11 (2.73)      -20.84 (2.43)   -27.07 (2.78)   -27.28 (2.79)   NS
  Lequesne\'s functional index                           -3.82 (0.51)       -4.80 (0.61)    -6.44 (0.59)    -5.39 (0.52)    \<0.05^†††^

^\*^Compared values of week 4 versus those of week 0 (run-in peroid), data represent mean (SEM). Statistic analysis: ^a^ = one-way ANOVA and Scheffe method. NS= no statistical significance. ^†^ = EA vs placebo and diclofenac group. ^††^ = combined vs placebo group. ^†††^ = EA vs placebo group.
:::

::: {#T5 .table-wrap}
::: {.caption}
######

Overall opinions of change and number of responders evaluated at week 4.
:::

  ------------------------------------ ------------------ --------------------- ------------- ------------------- -----------
                                       Treatment groups

  Parameter                            Placebo (n = 45)   Diclofenac (n = 49)   EA (n = 46)   Combined (n = 46)   *p*-value

  Orthopedist\'s overall opinion^\*^                                                                              0.01^†^
   Much better                         6                  18                    21            16
   Better                              22                 21                    20            23
   Same                                16                 10                    5             7
   Worse                               1                  0                     0             0

  Patient\'s overall opinion^\*^                                                                                0.09^†^
   Much better                         19                 25                    31            22
   Better                              16                 17                    11            23
   Same                                9                  7                     4             1
   Worse                               1                  0                     0             0

  Number of responders                 13                 18                    27            24                  0.02^††^
  ------------------------------------ ------------------ --------------------- ------------- ------------------- -----------

^\*^Data represent number of patients, ^†^ = chi-square test evaluated on the proportions of patients with the opinion of \"much better\", ^††^ = chi-square test.
:::

The percentage of patients who experienced adverse effects (e.g. gastrointestinal and central nervous system symptoms, rash, edema, and hypertension) during this study did not differ between the four groups (data not shown), whereas, local contusions around the knee were common in the EA and combined group (approximately 45%). However, the contusions usually disappeared within 5--7 days.

When the responders in each group were followed up for 2 months, the proportion of remaining responders was not significantly different between the four groups (Table [6](#T6){ref-type="table"}).

::: {#T6 .table-wrap}
::: {.caption}
######

Number of responders considered at the end of the study (week 4) and at 1 and 2 month(s) after treatment^\*^.
:::

                         1 month after treatment   2 months after treatment
  ----------------- ---- ------------------------- --------------------------
  Placebo           13   11/12                     9/12
  Diclofenac        18   10/16                     7/15
  EA                27   21/25                     19/24
  Combined          24   14/24                     14/24

  *p* value^\*\*^        0.12                      0.19

^\*^ Only the responders at the end of the study were followed up for 2 months. ^†^Some patients were unable to be evaluated due to loss of follow up or use of NSAIDs for other purposes during the follow-up period. ^\*\*^ Fisher test between the four groups.
:::

Discussion
==========

Although there are several lines of evidence from many controlled and uncontrolled studies for the short-term and long-term effectiveness of acupuncture in relieving clinical pain \[[@B23]-[@B28]\], the scientific data concerning the efficacy of acupuncture in OA are rare \[[@B19]\]. In addition, there are several systemic flaws \[[@B29]\] among these studies due to inadequate statistical power, inadequate sessions of acupuncture treatment, failure to control concomitant therapies, or no sham/placebo acupuncture controlled group. In this study, we minimized the methodological limitations of previous studies by using the randomized, single-blind, placebo controlled design with a larger sample size of OA patients coupled with standard outcome assessments. By using the percentage of the responders as the main efficacy criterion, the comparison between true and sham acupuncture needs at least 61 patients per group, whereas, only 35 patients per group are needed to compare between true and placebo acupuncture performed by not puncturing the skin \[[@B23],[@B30]\]. In order to increase the ability of differentiating true from placebo effects and minimize the sample size, we selected the procedure of attaching the acupuncture points with the patch electrodes as placebo EA, and at least 45 completers per group were treated in this trial. In this study, a double-blind design was considered inappropriate, since we used patch electrodes as placebo EA and our patients might have recognized the difference between true and placebo EA. A single blind was, therefore, a reasonable alternative.

The acupuncture points used were selected because we intended to determine only the effects of local points around the affected knee. This applied especially to the medial aspect, which related to the knee compartment that was frequently involved in OA. Using these local points coupled with the needling technique (developed by Chawal Kanchanakul, acupuncturist of Dhammanamai Foundation, Chiang Mai, Thailand) demonstrated a rather simple, convenient, less painful, and more acceptable method for Thai patients, and it was effective in our pilot study. The points selected here were therefore different from other trials \[[@B24]-[@B26],[@B28]\], which also included the distal points at medial and lateral aspects of the leg. Low-frequency (2 Hz) EA was selected because it produces an analgesia of long duration, which outlasts the 20-min stimulation session by 30 min to many hours. In addition, its effects are cumulative after several sessions of treatment given either daily or less frequently (2--3 times a week) \[[@B30]\]. For these reasons, the low-frequency EA in this study was therefore given 3 times a week for 4 weeks, as commonly recommended in EA practice. However, to balance the acupuncture point stimulation by positive and negative polarities, each point was stimulated 6 times with positive and negative polarities in an alternate sequence during 12 sessions of treatment. In addition, each pair of electrodes was connected to each pair of adjacent points in order to obtain equal electrical sensation in each point during stimulation.

In this study, the clinical responses observed in the placebo group might result from, 1) the placebo effect or natural fluctuations in the symptoms of OA that are unrelated to the analgesic effect of paracetamol, because some patients demonstrated reductions in these scores without or with minimal analgesic need, or 2) the direct effects of paracetamol as a rescue analgesic. The latter reason made the placebo group not absolutely inert because paracetamol is also the first-line drug in the treatment of OA of the knee. However, the use of a rescue analgesic could not be avoided due to ethical reasons.

At week 4, the improvement of symptom scores (except change in WOMAC pain index and WOMAC total score) and the number of responders/patients with the opinion of \"much better\" were greatest in the EA group. These data indicate the great potential of EA in the symptomatic treatment of OA of the knee. This study also demonstrated that EA was significantly more effective than placebo with respect to changes in VAS and Lequesne\'s functional index, and significantly more effective than diclofenac with respect to the changes in VAS. This superiority of EA indicates its genuine efficacy \[[@B17],[@B23],[@B30]\], which was more effective than placebo (or diclofenac) in this study. A previous trial revealed 34% and 14% reductions in the mean values of WOMAC total score and Lequesne\'s index, respectively after 4 weeks of acupuncture treatment \[[@B28]\], whereas, our study demonstrated a 50% and 45% reduction, respectively. These discrepancies might be due to differences in acupuncture point selection, number of points and the electrical stimulation technique used, or electrical stimulation parameters.

The clinical responses in the combined group were slightly superior to the diclofenac group, but not to the EA group (except for the tendency of being superior with respect to a change in the subscale of the standardized WOMAC: pain index). These responses indicate that EA may exert an adequate analgesic effect, a combination with diclofenac may not be of further benefit. Although a combination was not more efficacious than EA alone, it was significantly more so compared to placebo in terms of improvement in the WOMAC pain index, while EA was not. However, the nonsignificant change in clinical scores between the diclofenac and placebo group indirectly suggests that diclofenac may be as effective as paracetamol (as needed) in symptomatic treatment of OA of the knee.

The nonsignificant change in paracetamol consumption among the four groups during treatment might result from the unnecessary use of paracetamol in a great majority of active treatment groups, despite a significant improvement in some parameters of the EA and combined group, and a tendency to improve parameters in the diclofenac group. Another possibility might be the large variation in paracetamol consumption in each group that contributed to a false negative result. The nonsignificant change in the 50 feet-walk time among the four groups suggests that this parameter may not be sensitive enough to demonstrate existing differences. Walk time determined during stair climbing should thus be a better alternative \[[@B31]\]. The failure to demonstrate the differences in other parameters (i.e. change in WOMAC stiffness, disability, and total scores) between the active treatment and placebo group may be due mainly to the rescue analgesic and partly to inadequacy of the sample size.

This study demonstrated that EA treatment was safe and free from serious adverse effects. However, the indifference in adverse events between the diclofenac and placebo group might be due to the exclusion of patients who were at high risk to the adverse effects of NSAIDs during the screening visit or short-term trial. In this study, the rescue analgesic paracetamol contributed to several confounding effects. Therefore, the randomized placebo controlled trial without using rescue analgesic should be investigated further to confirm the effectiveness of this range of active treatment, especially in EA and its combination with NSAIDs.

In summary, EA was significantly more effective than placebo regarding reductions in 100 mm VAS and Lequesne\'s functional index, but was significantly more effective than diclofenac in only the reduction of 100 mm VAS. The combination of EA and diclofenac treatment was more effective than placebo with respect to the reduction in the subscale of standardized WOMAC (pain index), but not more effective than EA treatment alone. Local contusions were minor adverse effects commonly found in the EA and combined group. The positive effects far outweigh the serious adverse ones of EA, which make this procedure an attractive alternative treatment for patients with OA of the knee.

Competing Interests
===================

None declared.

Pre-publication history
=======================

The pre-publication history for this paper can be accessed here:

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Acknowledgements
================

This work was supported by the Faculty of Medicine, Chiang Mai University, Thailand.


Background
==========

Plants in the family Cruciferae, such as cabbage (*Brassica olerecea*), mustard (*Sinapis alba*) and crambe (*Crambe abyssinica*) produce glucosinolates which are secondary metabolites that can help plants defend themselves against phytophagous insects, fungi and other pests. Glucosinolates are hydrolyzed by thioglucosidase (myrosinase) enzymes to yield an aglycone which undergoes non-enzymatic rearrangements to produce organic isothiocyanates, thiocyanates, nitriles and other products \[[@B1],[@B2]\], although non-enzymatic thermal degradation of glucosinolates may produce chemical species identical to products of the enzymatic hydrolysis \[[@B3],[@B4]\]. The chemical structures of the hydrolysis products (breakdown products) of glucosinolates vary depending on the precursors in different plant species, and different endogenous or exogenous factors such as pH and ferrous ions \[[@B2],[@B5],[@B6]\]. Glucosinolates and their breakdown products have been the focus of many studies because of the possibility of using them as natural pesticides \[[@B7],[@B8]\]. However, it is considered that glucosinolates themselves possess limited biological activity until they are hydrolyzed \[[@B9]\]. The breakdown products are generally relatively small molecules which make many of them volatile. Many of these volatiles have been shown to act as attractants for certain insects seeking food or egg-laying sites rather than direct insecticidal activity \[[@B10]-[@B14]\]. Allyl glucosinolate (sinigrin) breaks down in soils to allyl isothiocyanate and allyl cyanide, and these breakdown products and glucosinolate-containing plant tissue have been suggested for use in controlling soil-borne plant pests \[[@B9],[@B15]-[@B19]\]. Despite the many efforts that have been devoted to exploring glucosinolates and their breakdown products as naturally occurring pesticides, few products have been marketed commercially. 2-(2-Butoxyethoxy)ethyl thiocyanate (Lethane 384^®^) and isobornyl thiocyanoacetate (Thanite^®^) were marketed as insecticides, and methylisothiocyanate (Vorlex^®^) as a fumigant, although their discovery may or may not have been directly related to glucosinolates. The thiocyanates (Lethane 384 and Thanite) were discontinued by their manufacturers in 1984 \[[@B20]\] and methylisothiocyanate, the closest relative of the glucosinolate breakdown products, was also banned in 1991 \[[@B21]\].

Harvested crops including seeds, grains and cereals suffer a loss of at least 10% from insect pests during storage. Losses of 30% are common throughout large areas of the world \[[@B22]\]. Many chemical fumigants such as ethylene dibromide and methyl bromide that have been used to control these stored grain insects are being banned or facing phase-out. There are clearly needs for alternative fumigants.

We have reported the insecticidal activity of the glucosinolates in crambe in our previous study, and found that allyl, 3-butenyl, 4-pentenyl, 4-hydroxybenzyl and 2-hydroxy-3-butenyl glucosinolate were the major glucosinolate components in crambe seed meal \[[@B8]\]. We also found that 2-hydroxy-3-butenyl glucosinolate was the predominant glucosinolate among the crambe glucosinolates, and its major breakdown product (\>95% of total glucosinolate) was 1-cyano-2-hydroxy-butene (CHB) \[[@B8]\]. CHB and other crambe glucosinolate breakdown products were found to have fumigation toxicity against insects in our early study \[[@B8]\].

Different fumigants have different insecticidal mode of actions, and some fumigants are known to affect the respiratory function of insects \[[@B20]\]. Although it has been reported that the CO~2~ expiration of *Tenebrio molitor* was reduced by feeding glucosinolate-containing rapeseed meal \[[@B24]\], there are no previous studies on the effect of the glucosinolate breakdown products on insect respiration.

In this paper we report the insecticidal activity of crambe-related glucosinolate breakdown products as fumigants and the effect of these chemicals on the respiratory function of insects. The latter is achieved by measuring the CO~2~ emission of the insects that are exposed to the chemicals. The fumigation effects of the breakdown products were compared and discussed together with the known fumigants dichlorovos and chloropicrin. The effect of the glucosinolate breakdown products on CO~2~ emission was also compared with that of hydrogen cyanide and rotenone.

Results and Discussion
======================

The house fly (*Musca domestica*) and the lesser grain borer (*Rhyzopertha dominica*) were chosen in the laboratory toxicity test, based on the availability and their significance as pests. In fact, glucosinolate-containing extracts from crambe were found to be toxic to the larvae of the house fly \[[@B8]\]. In the present study, we have found that some of the individual glucosinolate breakdown products from crambe were insecticidal toward *M. domestica* as potent fumigants. The respective LC~50~ of allyl thiocyanate (ATC), allyl isothiocyanate (AITC) and allyl isocyanate(AIC) were 0.1, 0.13 and 0.63 μg cm^-3^ (Table [1](#T1){ref-type="table"}). This is in the same potential range of the toxicity of chloropicrin (0.08 μg cm^-3^), a commercial fumigant. CHB, the major breakdown product of crambe glucosinolates was not as potent a fumigant as others. Its LC~50~ was 6.2 μg cm^-3^ (Table [1](#T1){ref-type="table"}).

::: {#T1 .table-wrap}
::: {.caption}
######

Fumigation toxicity of glucosinolate breakdown products against the house fly adults in comparison with selected fumigants (exposed for 24 h).
:::

  compound                     LC~50~ (μg cm^-3^)   95% FL
  ---------------------------- -------------------- --------------
  dichlorovos                  \<0.02               --
  chloropicrin                 0.08                 0.076--0.099
  allyl thiocyanate            0.1                  0.08--0.12
  allyl isothiocyanate         0.13                 0.10--0.16
  allyl isocyanate             0.63                 0.56--0.70
  allyl cyanide                3.66                 3.11--4.10
  1-cyano-2-hydroxy-3-butene   6.2                  4.91--9.73
  control                      --                   --
:::

Different insects have different susceptibilities toward chemicals. Although dichlorvos was almost 10 times as toxic as chloropicrin in house fly test (Table [1](#T1){ref-type="table"}), it was only 5 times more toxic in the lesser grain borer test (Table [2](#T2){ref-type="table"}). ATC, the most potent fumigant against the house fly was the most toxic against the lesser grain borer as well. Its LC~50~, 0.55 μg cm^-3^, against the latter insect was between that of dichlorvos (0.29 μg cm^-3^) and chloropicrin (1.3 μg cm^-3^). LC~50~\'s of allyl isothiocyanate (1.57 μg cm^-3^) was in the same range of toxicity as chloropicrin. Allyl isocyanate (2.2 μg cm^-3^) and allyl cyanide (2.8 μg cm^-3^) were slightly less toxic compared to chloropicrin, although they were all about 1/10 as toxic as dichlorvos (Table [2](#T2){ref-type="table"}) Allyl isocyanate was tested to compare with the glucosinolate breakdown products.

::: {#T2 .table-wrap}
::: {.caption}
######

Fumigation toxicity of glucosinolate breakdowns products against the lesser grain borer in comparison with selected fumigants (exposed for 24 h).
:::

  compound                     LC~50~ (μg cm^-3^)   95% FL       CO~2~^\*^
  ---------------------------- -------------------- ------------ -----------
  dichlorovos                  0.29                 0.21--0.41   --
  chloropicrin                 1.3                  1.20--1.42   --
  allyl thiocyanate            0.55                 0.50--0.60   5.1
  allyl isothiocyanate         1.57                 1.47--1.67   5.1
  allyl cyanide                2.8                  2.26--3.48   --
  1-cyano-2-hydroxy-3-butene   \>19.60              --           --
  allyl isocyanate             2.2                  2.06--2.34   10.4
  hydrogen cyanide             --                   --           4.2^\*\*^
  control                      --                   --           2.7
  control (HCN)                --                   --           4.6^\*\*^

^\*^ CO~2~ in μg/mg body weight by *B. germanica.* Exposed for 1 h. ^\*\*^ Done in a separate experiment.
:::

The high fumigant toxicity of the glucosinolate breakdown products against the lesser grain borer may have important economic impact on stored grain insects management. These naturally occurring chemicals were also insecticidal as fumigant on other stored grain insects such as the sawtoothed grain beetle (*Oryzeaphilus surinamensis* L.) and the red flour beetle (*Tribolium castaneum* Herbst) (Tsao, et al., unpublished data, 1997).

Long-term fumigation tests against the lesser grain borer showed little improvement in the toxicity of the glucosinolate breakdown products. Also, the 2-day and 4-day tests resulted in higher mortality in the control (data not shown).

Little is known about the mode of action underlying the high fumigation toxicity of the glucosinolate breakdown products. Dichlorvos exerts its insecticidal toxicity against the insect nervous system, by inhibiting acetylcholinesterase. Methyl bromide and chloropicrin\'s insecticidal mode of action has been shown as their reactions with the nucleophilic sites such as OH, SH or NH~2~ groups in vital enzymatic systems \[[@B25]\]. Many non-fumigant insecticides are known to affect the respiratory function of insects (e. g., rotenone, dinitrophenols) \[[@B20]\]. In our fumigation test technique, inhalation (intake through the spiracles) was the only way for the insects to receive the volatile chemicals, although certain degree of absorption could occur through the cuticle. Monitoring the CO~2~ production of the insects at sub-lethal doses in a closed chamber may provide valuable symptomatic information toward understanding how these chemical volatiles affect the respiration of insects and lead to the eventual death of the insects. Monitoring CO~2~ emission is one of the well established technique to study insect respiration and the effect of chemicals on the respiratory function, although it has sometimes been accompanied with simultaneous monitoring of oxygen consumption \[[@B24],[@B26]\].

The quantity of the chemicals used in our CO~2~ test was based on the toxicity tests of the chemicals (data not shown). At the sub-lethal levels, no mortality was observed within the test period. For AIC, ATC and AITC, the amount of chemical utilized was 200 μl of 10^4^ ppm corn oil solution. This is equivalent to ca. 10 μg cm^-3^ of the chemical in air. This concentration is lethal to the lesser grain borer and the house fly, but not to the cockroaches due to different physiological aspects and the shorter exposure time in the CO~2~ monitoring test.

By exposing *B. germanica* to the fumes of the glucosinolate breakdown products AIC, ATC and AITC, we found that the CO~2~ production of this insect was significantly increased at each time interval (Tables [1](#T1){ref-type="table"} and [2](#T2){ref-type="table"}, Fig. [2](#F2){ref-type="fig"}). AIC was the strongest CO~2~ emission (respiration) enhancer. *B. germanica* treated with AIC produced more than 3 times more CO~2~ than that in the control after being exposed for 1 h and 2 h. Even after 4 h, the CO~2~ emission was more than 75% higher for AIC (Fig. [2](#F2){ref-type="fig"}). Although ATC and AITC did not increase the CO~2~ emission as strongly as did AIC, these two chemicals still showed significant effects after 2 h. The effect was not as significant at 4 h for ATC and AITC (Fig. [2](#F2){ref-type="fig"}).

![The effect of AIC, ATC and AITC on the CO~2~ emission of the *B. germanica* adults at different time intervals. Bars within the same group followed by the same letter are not significantly different (LSD, α = 0.05).](1472-6785-2-5-2){#F2}

HCN and rotenone were tested for comparing the mode of action of glucosinolate breakdown products, because they are potential inhibitors of CO~2~ production \[[@B20]\]. AITC may have a different mode of action on the respiratory system than HCN and rotenone as the CO~2~ production of *P. americana* was significantly increased by AITC as compared to the control (Fig. [3](#F3){ref-type="fig"}). The CO~2~ emission of *P. americana* was not significantly affected by HCN or rotenone during the test period (Fig. [4](#F4){ref-type="fig"} and [5](#F5){ref-type="fig"}), indicating that these chemicals may not affect the respiratory system of the insect under the dose used in this experiment. The less effectiveness could be due in part to the low concentration of HCN and rotenone. Effort to use extended exposure time was also tried, however, high morality was observed when *P. americana* was exposed to HCN and rotenone at higher dose, or for longer time (\>60 min and \>35 min, respectively). There was no particular reason except availability for using *P. Americana* instead of *B. germanica.*

![The effect of AITC on CO~2~ emission of the *P. americana* at different time intervals. Each data point is an average of two replications.](1472-6785-2-5-3){#F3}

![The effect of hydrogen cyanide on CO~2~ emission of the *P. americana* nymphs at different time intervals. Each data point is an average of two replications.](1472-6785-2-5-4){#F4}

![The effect of rotenone on CO~2~ emission of the *P. americana* at different time intervals. Each data point is an average of two replications.](1472-6785-2-5-5){#F5}

Toxicity of isothiocyanates has been attributed to inactivation of the thiol group of essential enzymes \[[@B27]\]. The mode of action of many isothiocyanate compounds has also been attributed to their capability for alkylating the nucleophilic groups of biopolymers such as DNA, thus having cytotoxic properties which can affect the formation of the spiracular epidermis and crochet on the prolegs of tobacco hornworm (*Manduca sexta* L.) caterpillars \[[@B28]-[@B30]\]. Our bioassay avoids totally any direct contact between the insects and the chemical, thus the insecticidal effect is very likely caused by inspiration of the chemical by the insects through their spiracles. The increased CO~2~ emission observed in this study indicated a different mode of action of these glucosinolate breakdown products. This new mode of action may be similar to that of the dinitrophenols, although further investigation is needed to prove this speculation. Compounds such as dinitrophenol are uncouplers or inhibitors of oxidative phosphorylation \[[@B31]\]. When the coupling between the respiratory chain and phosphorylation is broken, respiratory control is lost, and the electron transport along the chain occurs at full speed without producing ATP, the high energy molecule. Uncouplers kill insects not by inhibiting normal electron transport of the respiratory chain, but rather by enhancing the respiration \[[@B27],[@B31]\]. When the energy-transfer (production of ATP) is blocked, and at the same time, the respiration is accelerated, the insects have to use their stored energy sources and the exhaustion of those resources then lead directly to the death of the insects \[[@B31]\]. Insecticides such as rotenone and HCN are known to affect the respiratory function by inhibiting certain enzymes in the respiratory electron transport chain of insects, thus reducing the oxygen consumption and perhaps CO~2~ production as well \[[@B20]\]. This may explain why HCN did not increase the CO~2~ emission at low dose.

As with many other conventional fumigants, we also found that some of the glucosinolate breakdown products and related chemicals were strong hatching inhibitors for the soybean cyst nematode (*Heterodera glycines*) (unpublished data, 1996)

Conclusions
===========

Using glucosinolate breakdown products as safer fumigants in pest control is a novel approach in integrated pest management, and at the same time, since these natural products are considered fully biodegradable and less toxic, considering many cruciferous vegetables are consumed daily, there should be fewer environmental concerns. The bioactivity of these compounds may be from their effect on the insect respiratory system indicated by the inhibited or accelerated CO~2~ production.

Materials and Methods
=====================

Chemicals
---------

Dichlorovos and chloropicrin were purchased from ChemService (West Chester, PA). Allyl isothiocyanate, allyl isocyanate, allyl cyanide and rotenone were purchased from Aldrich Chemical Co. (Milwaukee, WI). Allyl thiocyanate was synthesized in this laboratory by reacting allyl chloride with potassium thiocyanate (KSCN) in dimethyl sulfoxide (DMSO). Briefly, KSCN (0.31 mol) and DMSO (200 ml) were added to a 1-L flask and heated on a water bath to 85°C followed by addition of allyl chloride (0.30 mol) to the flask. The mixture was allowed to cool to room temperature, and then the product ATC was extracted with ethyl ether (200 ml × 4). ATC was purified using column chromatography on silica gel with 2/10 ether/hexane, and the structure was confirmed by nuclear magnetic resonance (NMR) and infrared (IR) spectroscopy: ^1^H NMR δ~TMS~ (CDCl~3~) ppm: 3.58 (2H, d, -C[H]{.ul}~2~-SCN), 5.34--5.43 (2H, dd, C[H]{.ul}~2~-CH-), 5.86--6.02 (1H, m, CH~2~-C[H]{.ul}-); IR: 2930--2960, 2870, 2160, 1640,1400, 1230, 985, 930 cm^-1^. l-Cyano-2-hydroxy-3-butene (CHB) was extracted from the defatted crambe meal. One hundred g of the crambe meal (National Sun Inc., Enderland, ND) was extracted with CH~2~Cl~2~ at 45°C in a Soxhlet apparatus overnight, and isolated and purified by using column and thin layer chromatography. The structure of CHB was confirmed by NMR and mass spectrometry: ^1^H NMR δ~TMS~ (CDCl~3~) ppm: 2.60 (2H, dd, -C[H]{.ul}~2~-CN), 2.75 (H, brs, -CH(O[H]{.ul})-), 4.37--4.50 (1H, m, -C[H]{.ul}(OH)-), 5.25--5.45 (2H, m, C[H]{.ul}~2~-CH-), 5.85--5.98 (1H, m, CH~2~-C[H]{.ul}-); IR: 3480, 3000, 2930, 2270, 1640, 14251135, 1070, 1000, 945 cm^-1^. The above NMR was consistent with that of Das and Torssell \[[@B32]\] and the IR data is in accordance with Daxenbichler *et al.*\[[@B33]\].

Fumigation tests
----------------

### Fumigation of the House Fly

Approximately 1 g 1/1 dry sucrose/powdered milk was placed in the bottom of a 40-ml jar, along with a 2-cm piece of cotton roll moistened with distilled water. Ten to 15 house fly adults (*Musca domestica* L., laboratory reared Orlando regular strain, one week post-emergence) were anesthetized with carbon dioxide and placed in the 40-ml jars. The jars were each covered with nylon mesh secured by a rubber band. Three 40 ml jars were placed in the bottom of a 2745 ml amber jar. A filter paper (Whatman No. 1, 9-cm diameter) was folded in quarters and placed in the jar as well. About 30 min after the flies recovered from anesthesia, 200 μl of the appropriate corn oil (Flavorite™ pure corn oil, Preferred Products, Inc., Chaska, MN) solution of the chemicals was applied to the filter paper with an eppendorf pipette, the disposable tip of which was left in the jar. The control was treated under the same conditions with a corn oil blank. The above jar was tightly sealed and left for 24 h, when mortality was read. The insects were considered dead when they displayed no observable motion when prodded. LC~50~ was calculated based on 5 concentrations.

### Fumigation of the lesser grain borer

Ten adult lesser grain borers (*Rhyzopertha dominica* Fabricius, laboratory reared Manhattan KS strain) were placed in a 1.5 × 5-cm glass tube with 2 g whole wheat kernels. The tubes were closed on both ends by a tight metal mesh secured with parafilm. Three tubes were tied together with a wire, and suspended from the top of a jar (490 ml). A filter paper (same as used in the fly test), was folded in quarters and placed at the bottom of the jar. One hundred μl of the appropriate corn oil dilution of the chemicals was applied to the filter paper with an eppendorf pipette, the disposable tip of which was left in the jar. Corn oil was used in the control. The jar was tightly capped and left for 24 h, at which time the mortality was recorded. An insect was considered dead when it displayed no observable motion upon prodding. LC~50~ was calculated based on 5 concentrations.

### Measuring CO~2~ emitted by insects

We chose the German (*Blattella germanica* L.) and the American cockroach (*Periplaneta americana* L.) in this test because they are non-flying, thus producing a steady and measurable quantity of CO~2~. Two adult *B. germanica* (1 male, 1 female without ootheca) or 2 nymphs (average weight 0.55 g) of *P. americana* were weighed and put in a glass tube (5 × 1.5 cm) with both ends covered with mesh screen. A bigger size of tubing (5 × 2 cm) was used for the *P. americana.* The tube was then placed into a glass bottle (220 ml) together with a quarterly folded filter paper (same as used above). A rubber stopper with glass inlet and outlet was tightly capped on the bottle with both ends sealed with a rubber septum (Fig [1](#F1){ref-type="fig"}). An infrared carbon dioxide analyzer (LIRA^®^ Infrared gas analyzer, Model 3000, Mine Safety Appliances Company, Pittsburgh, PA) was used to measure the emitted CO~2~. The sealed bottle was flushed with CO~2~-free air (compressed air filtered through soda lime tubes and a NaOH trap) before introducing the chemical. A sub-lethal dose of the breakdown products (200 μl of 10000 ppm chemical in corn oil for the *B. germanica,* and 100 μl of 1000 ppm for *P. americana*) was immediately injected with a syringe through the septum onto the filter paper inside the system (Fig. [1](#F1){ref-type="fig"}). In this system, direct contact between the insects and the chemical is completely avoided. The CO~2~ emitted by the test insects was recorded at Range 1 (the most sensitive range) of the instrument at different exposure times. There were three replicates for each treatment unless stated otherwise, and the same volume of corn oil was used in the control. Tests on the effect of HCN were achieved by wetting the filter paper with 100 μl of 1 N HCl, and, after flushing out the CO~2~, injecting 20 μl of 1 N NaCN aqueous solution onto the paper. Rotenone (5 μl of 100 ppm in acetone) was injected to the thorax of the *P. americana* nymph in the CO~2~ test and same the amount of acetone was used in the control. Water or acetone was used in the control for HCN or rotenone.

![A descriptive diagram of the apparatus used in the insect CO~2~ monitoring test.](1472-6785-2-5-1){#F1}

The recorded peak meter readings (PMR) were converted to mg of CO~2~ according to the following formula \[[@B34]\]:

CO~2~ (mg) = 0.0092+0.00795\*PMR+0.0000002\*(PMR)^2^

Data analysis
-------------

Probit analysis was used to determine LC~50~, 95% FL, and slopes according to methods outlined by Finney \[[@B35]\]. Means were separated using least significant differences (LSD) (P = 0.05) \[[@B36]\].

Acknowledgements
================

The authors thank Dr. T. A. Anderson of the Texas Tech University, Texas, USA for his assistance in the use of the infrared carbon dioxide analyzer.


Background
==========

Transforming growth factor beta (TGF-β) family proteins are multifunctional cytokines capable of regulating cell growth, extracellular matrix protein synthesis, and immune cell functions \[[@B1],[@B2]\]. Hyaluronidase is an extracellular matrix-degrading enzyme. Interestingly, malignant and metastatic breast and prostate cancer cells frequently overexpress both hyaluronidase and TGF-β proteins \[[@B3]-[@B7]\]. Previously we have shown that bovine testicular hyaluronidase, also known as PH-20, increases the susceptibility of various cancer cells to tumor necrosis factor (TNF or TNF-α) cytotoxicity \[\[[@B8]\], review\]. PH-20 induces the expression of proapoptotic p53 \[[@B9]\] and WOX1 (WW domain-containing oxidoreductase; also known as WWOX or FOR) \[[@B10]\], which contributes to the increased TNF cytotoxic effect. PH-20-induced downregulation of the extracellular matrix inter-α-inhibitor is also associated with the enhanced TNF cytotoxicity \[[@B9]\].

TGF-β family proteins, including β1,β2 and β3, induce TNF-resistance and suppress the PH-20 effect of increasing TNF cytotoxicity in murine L929 fibroblasts \[[@B8],[@B11]\]. TGF-β1 induces a novel extracellular matrix protein that prevents TNF-mediated cell death and blocks the activation of extracellular signal-regulated kinase (ERK; also known as p42/44 mitogen-activated protein kinase, p42/44 MAPK) in L929 cells \[[@B8],[@B12]\]. Additionally, TGF-β1 induces the expression of TIAF1 (TGF-β-induced antiapoptotic factor) \[[@B13]\] and TIF2 (TGF-β-induced factor 2) \[[@B14]\] that inhibit TNF cytotoxicity. Whether these TGF-β1-induced proteins restrict the ability of PH-20 to increase TNF cytotoxicity is unknown.

PH-20 blocks TGF-β1-mediated growth suppression of epithelial cells \[[@B8],[@B15]\]. Additionally, PH-20 rapidly activates ERK in L929 cells and TGF-β1 reduces the PH-20-induced ERK activation \[[@B16]\]. In contrast, TGF-β1 synergistically increases PH-20-mediated inhibition of staurosporine apoptosis \[[@B16]\]. Thus, PH-20 and TGF-β1 are physiological antagonists for only certain cellular responses.

Hyaluronidases Hyal-1 and Hyal-2 play a critical role in cancer invasion and metastasis \[[@B3]-[@B5]\], although inactivation of *HYAL1* gene has been shown in head and neck squamous cell carcinomas \[[@B17]\]. Hyal-1, also known as Luca-1, is a lysosomal enzyme that is secreted from cells. Hyal-1 is a candidate tumor suppressor \[[@B5],[@B18]\], although it enhances extravasation and metastasis of prostate cancer cells \[[@B3]\]. Also, mutations in Hyal-1 may contribute to the pathogenesis of a lysosomal disorder, mucopolysaccharidosis IX \[[@B19]\]. Hyal-2 is a lysosomal protein, which is active at low pH \[[@B20]\]. This protein is also known to be a glycosylphosphatidylinositol (GPI)-anchored cell-surface receptor for jaagsiekte sheep retrovirus \[[@B21]\].

Testicular hyaluronidase PH-20 counteracts TGF-β1-induced TNF-resistance in L929 cells \[[@B8]\]. The goal of the present study was to examine whether lysosomal Hyal-1 and Hyal-2 enhance TNF cytotoxic function and counteract TGF-β1-mediated TNF-resistance in L929 cells. It was determined that stable expression of murine Hyal-2 in L929 cells increased their sensitivity to TNF-mediated death, whereas Hyal-1 was somewhat less effective. TGF-β1 blocked this increased sensitivity. In contrast, Hyal-1 and Hyal-2 did not enhance L929 cell death by anticancer drugs such as daunorubicin, actinomycin D, staurosporine and camptothecin. Overexpressed Hyal-1 and Hyal-2 induced the expression of proapoptotic WOX1, but not p53, suggesting a role of WOX1 in the increased cellular susceptibility to TNF cytotoxicity. Additional mechanisms that are associated with the increased TNF cytotoxicity were also investigated.

Results
=======

As summarized in Figure [1](#F1){ref-type="fig"}, PH-20 induces the expression of proapoptotic p53 and WOX1 \[[@B9],[@B10]\] and downregulates antiapoptotic matrix inter-α-inhibitor \[[@B9]\], thereby enhancing TNF susceptibility in L929 cells. Also, PH-20 rapidly activates ERK and c-Jun *N*-terminal kinase (JNK1 and JNK2) \[[@B16]\]. Whether these signaling events lead to induction of p53 and WOX1 is unknown.

![Proposed basis for the antagonistic actions of PH-20 and TGF-β1 on L929 cell susceptibility to TNF cytotoxicity. Testicular hyaluronidase PH-20 enhances TNF cytotoxicity in L929 fibroblasts (see green), whereas TGF-β1 induces TNF-resistance in these cells (see red). Both upregulation of proapoptotic p53 and WOX1 and downregulation of antiapoptotic matrix inter-α-inhibitor are involved in the PH-20-increased TNF susceptibility in L929 cells \[[@B8]-[@B10]\]. Also, PH-20 rapidly activates ERK (or p42/44 MAPK) and c-Jun *N*-terminal kinases (JNK1 and JNK2) \[[@B16]\]. In contrast, TGF-β1 protects L929 cells from TNF cytotoxicity by upregulating antiapoptotic TIAF1, TIF2 and a novel matrix protein \[[@B11]-[@B14]\]. TGF-β1 counteracts PH-20-induced TNF-susceptibility in L929 cells \[[@B8]\]. TGF-β1 suppresses PH-20-mediated ERK activation \[[@B16]\], whereas ERK activation is not related with PH-20-increased TNF susceptibility in L929 cells \[[@B15]\]. Whether the proapoptotic p53 and WOX1 block the antiapoptotic function of TIAF1 and TIF2 is not known.](1471-2121-3-8-1){#F1}

In contrast, TGF-β1 protects L929 cells from TNF cytotoxicity by upregulating the expression of antiapoptotic TIAF1, TIF2 and a novel matrix protein \[[@B11]-[@B14]\]. TGF-β1 blocks PH-20-increased TNF susceptibility \[[@B15]\]. Likewise, PH-20 inhibits TGF-β1-induced TNF-resistance \[[@B15]\]. TGF-β1 suppresses PH-20-mediated ERK activation \[[@B16]\]; however, the ERK activation is not related with PH-20-increased TNF susceptibility in L929 cells \[[@B15]\]. Whether the proapoptotic p53 and WOX1 block the function of antiapoptotic TIAF1 and TIF2 is not known. In this study, experiments were designed to determine whether lysosomal Hyal-1 and Hyal-2 enhance TNF cytotoxic function and counteract TGF-β1-mediated TNF-resistance in L929 cells.

The full-length murine hyaluronidase *Hyal1* and *Hyal2* cDNAs were found in the EST database (GenBank accession AA688635 and BF139787, respectively) and the determined DNA sequences have been deposited in the GenBank (*Hyal1*, AF422176; *Hyal2*, AF422177). The *Hyal1* cDNA encodes a 463-amino-acid protein, which has 99.1% identity to a reported murine sequence (AF011567) \[[@B18]\]. Differences in the protein sequences are Gly-247, Gly-284 and Cys-450 in our Hyal-1 protein, compared to Arg-247, Glu-284 and Arg-450 in AF011567. Transitions in these amino acid residues are non-conservative.

The murine *Hyal2* cDNA (AF422177) encodes a 473-amino-acid protein. The deduced protein sequence is identical to a reported murine sequence (AF302844), but has an Ile-355 to Val-355 transition compared to another clone (AF302843). Variation in the protein sequence is also found in a murine Hyal-2 homologue (NP_034619) \[[@B22]\], which possesses three unique 2-, 5- and 7-amino-acid segments, not identical to the above Hyal-2 proteins.

L929 cells were engineered to stably express GFP-Hyal-1 or GFP-Hyal-2 fusion proteins. The presence of these proteins in these cells was demonstrated in Western blotting using anti-GFP antibody (data not shown). Exogenous bovine testicular hyaluronidase PH-20 stimulates rapid activation of c-Jun *N*-terminal kinases (JNK1 and JNK2) in L929 cells \[[@B16]\]. In contrast, the stably expressed Hyal-1 and Hyal-2 could not mediate constitutive activation of JNK1 and JNK2 in L929 cells (data not shown).

Exposure of the Hyal-2-expressing L929 cells to TNF for 16--24 hr resulted in increased cell death by \~60--110%, compared to the control GFP-expressing cells (Fig. [2](#F2){ref-type="fig"}). These results are similar to the bovine testicular PH-20-increased TNF cytotoxicity of L929 cells \[[@B15]\]. In contrast, there were no significant differences in the extent of cell death when GFP-Hyal-2 and control GFP-expressing cells were exposed to staurosporine, actinomycin D, daunorubicin, or camptothecin for 16--24 hr (Fig. [2](#F2){ref-type="fig"}).

![Hyal-2 increases L929 cell sensitivity to TNF cytotoxicity. L929 cell lines that stably expressed GFP and GFP-Hyal-2 were established (see Materials and Methods) and exposed to TNF-α, staurosporine, actinomycin D (ActD), daunorubicin, or camptothecin for 24 hr. The extent of cell death were measured by staining the cells with crystal violet as previously described (mean ± standard deviation; n = 8) \[[@B10]\].](1471-2121-3-8-2){#F2}

There was also an increased susceptibility to TNF cytotoxicity in the Hyal-1-expressing L929 cells (approximately 20--90% increase; also see Fig. [7](#F7){ref-type="fig"}), compared to the GFP control cells. However, there were no significant differences among these cells in the extent of death caused by staurosporine, actinomycin D, daunorubicin and camptothecin (data not shown).

In similar experiments, the effect of transient expression of Hyal-1 and Hyal-2 on cellular sensitivity to anticancer drugs was examined. L929 cells were electroporated with the GFP-Hyal-1 or GFP-Hyal-2 construct, cultured for 48 hr and then exposed to actinomycin D, daunorubicin or camptothecin for 24 hr. There were no differences in cell death in these Hyal-1 and Hyal-2-expressing cells, compared to control cells (data not shown). These findings are consistent with the results for stable transfectants presented above (Fig. [2](#F2){ref-type="fig"}).

Bovine testicular PH-20 enhances TNF sensitivity in L929 cells by increasing the expression of proapoptotic p53 and WOX1 \[[@B9],[@B10]\]. The ability of Hyal-1 and Hyal-2 to induce the expression of p53 and WOX1 in L929 cells was examined. L929 cells were electroporated with nothing (null), GFP, GFP-Hyal-1 or GFP-Hyal-2 constructs and cultured for 48 hr. Transient expression of GFP-Hyal-1 or GFP-Hyal-2 in L929 cells induced WOX1 expression, whereas no WOX1 expression was observed in non-transfected cells or control GFP-expressing cells (Fig. [3A](#F3){ref-type="fig"}). The 46-kDa band is full-length WOX1, while the 31-kDa band is a degraded WOX1 \[[@B10]\]. No detectable p53 expression was observed in these cells (Fig. [3A](#F3){ref-type="fig"}).

![Hyal-2 induces WOX1 expression in L929 cells. (**A**) L929 cells were electroporated with nothing (null), GFP, GFP-Hyal-1 or GFP-Hyal-2 construct (30 g DNA / 3 × 10^6^ cells) and cultured for 48 hr. Transient expression of GFP-Hyal-1 and GFP-Hyal-2 in L929 cells induced WOX1 expression (46 and 31 kDa), whereas no WOX1 expression was observed in non-transfected cells or control GFP-expressing cells. The 46-kDa band is full-length WOX1, and the 31-kDa band is a degraded WOX1 \[[@B10]\]. No detectable p53 expression was observed in these cells. α-Tubulin was examined to confirm equal protein loading onto each lane of the gel. (**B**) In comparison, the GFP-Hyal-2 stable transfectant constitutively expressed cytosolic WOX1 (46 kDa) and TNF (50 ng/ml) reduced the WOX1 expression. The reduction is probably due to TNF-mediated WOX1 nuclear translocation from the mitochondria \[[@B10]\]. In contrast, WOX1 was not expressed in the stable GFP-Hyal-1 cells, and TNF induced the expression of WOX1 in a time-related manner. TNF could not induce WOX1 expression in the control GFP-expressing cells. (**C**) L929 cells were transfected with GFP-Hyal-2 and/or WOX1 constructs by electroporation. GFP-Hyal-2 enhanced WOX1-mediated L929 cell death, as determined 48 hr post-transfection using crystal violet staining (two representative rows of cell stains shown at left and the analyzed data shown at right; mean ± standard deviation, n = 4). cell death. In controls, GFP-Hyal-1 or GFP-Hyal-2 alone failed to induce cell death.](1471-2121-3-8-3){#F3}

In comparison, the cytosolic WOX1 (46 kDa) was constitutively expressed in the L929 cells stably transfected with the GFP-Hyal-2 construct, while TNF reduced WOX1 expression in a time-dependent manner (Fig. [3B](#F3){ref-type="fig"}). This reduction is probably due to TNF-mediated WOX1 nuclear translocation from the mitochondria \[[@B10]\]. In contrast, WOX1 was not expressed in L929 cells stably transfected with the GFP-Hyal-1 construct, and TNF induced WOX1 expression in 40 min (Fig. [3B](#F3){ref-type="fig"}). TNF did not induce WOX1 expression in the control GFP-expressing cells (Fig. [3B](#F3){ref-type="fig"}).

Whether Hyal-2 enhances the apoptosis-inducing function of WOX1 was determined. In transient transfection experiments, L929 cells were electroporated with GFP-Hyal-2 and/or WOX1 constructs. GFP-Hyal-2 enhanced WOX1-mediated L929 cell death, as determined 48 hr post-transfection using crystal violet staining (Fig. [3C](#F3){ref-type="fig"}). In contrast, GFP-Hyal-1 did not enhance WOX1-mediated cell death (Fig. [3C](#F3){ref-type="fig"}). GFP-Hyal-1 and GFP-Hyal-2 alone did not mediate cell death (Fig. [3C](#F3){ref-type="fig"}).

Next, the effect of TNF-mediated IκBα degradation and NF-κB activation was examined in both Hyal-1 and Hyal-2 cells. The GFP-Hyal-1 and GFP-Hyal-2 stable transfectants and control cells were exposed to TNF for various times. IκBα degradation and NF-κB activation (or NF-κB nuclear translocation) were observed at 20 to 60 min in Hyal-2 cells post TNF stimulation, but at the 20 min time point in both control and Hyal-1 cells (Fig. [4](#F4){ref-type="fig"}).

![TNF mediates a prolonged period of IκBα degradation and NF-κB activation in L929 cells stably expressing GFP-Hyal-2. The GFP-Hyal-1 and GFP-Hyal-2 stable transfectants, as well as control L929 cells, were exposed to TNF (50 ng/ml) for 0, 20, 40, 60 and 120 min, followed by analysis of protein expression by Western blotting. TNF mediated a prolonged cytosolic IκBα degradation, as well as a nuclear presence of NF-κB p65 (or NF-κB activation), over a 20 to 60 min time period in Hyal-2 cells. A similar effect was seen only at 20 min in Hyal-1 and control cells.](1471-2121-3-8-4){#F4}

To further understand how Hyal-2 participates in the apoptosis pathway, the translocation of Hyal-2 to the mitochondria was examined during cell death. Transient expression of GFP-Hyal-2 in COS7 fibroblasts showed the localization of the expressed protein in the lysosome but not in the mitochondria, as determined by staining the cells using LysoTracker and Mitotracker Red (data not shown). These observations are consistent with other reports \[[@B5],[@B20]\]. COS7 cells are TNF-resistant. The reasons for using this cell type are its large size (which provides better resolution under fluorescence microscopy) and the ease of transfection and gene expression using liposome-based reagents such as FuGene 6 and GeneFector. Exposure of the GFP-Hyal-2-expressing COS7 cells to staurosporine (for inducing apoptosis) resulted in a time-related increase in co-localization of GFP-Hyal-2 with the mitochondria (Fig. [5](#F5){ref-type="fig"}), suggesting that a portion of GFP-Hyal-2 migrates to the mitochondria during apoptosis.

![Migration of Hyal-2 to the mitochondria during staurosporine-mediated apoptosis. COS7 cells were transfected with the GFP-Hyal-2 construct using FuGene 6 (Roche) or GeneFector (Venn Nova). The cells were cultured for 24--48 hr and then treated with staurosporine (stauro). Mitochondria (Mito) were stained with Mitotracker Red and nuclei were stained with DAPI. Co-localization analysis showed that there was a time-related increase in co-localization of GFP-Hyal-2 with mitochondria (see merged), suggesting that a portion of GFP-Hyal-2 migrates to the mitochondria during apoptosis. The nuclear stain is shown at the 2- and 4-hr time points only.](1471-2121-3-8-5){#F5}

Next, whether TGF-β1 antagonizes the function of Hyal-1 and Hyal-2 in growth regulation was examined. TGF-β1 inhibits epithelial cell growth but promotes fibroblast growth \[[@B15]\]. TGF-β1 inhibition of epithelial cell growth is blocked by bovine testicular PH-20 \[[@B15]\]. In agreement with our previous observations \[[@B11]\], TGF-β1 promoted L929 cell growth by approximately 20% in 24--48 hr (Fig. [6](#F6){ref-type="fig"}). However, TGF-β1 did not promote the growth of L929 cells stably expressing Hyal-1 or Hyal-2 (Fig. [6](#F6){ref-type="fig"}).

![TGF-β1 promotes L929 cell growth but not the growth of Hyal-1 and Hyal-2-expressing L929 cells. Non-transfected L929 cells or the stable GFP-Hyal-1 and GFP-Hyal-2-expressing cells were treated with TGF-β1 (2 ng/ml) for 24 hr. The extent of cell growth was measured by staining the cells with crystal violet (mean ± standard deviation; n = 8).](1471-2121-3-8-6){#F6}

Finally, the functional antagonism between TGF-β1 and Hyal-1 or Hyal-2 in regulating TNF susceptibility in L929 cells was examined. The Hyal-1 and Hyal-2 stable transfectants were pretreated with TGF-β1 for 1 hr and then treated with TNF overnight. Both Hyal-1- and Hyal-2-increased TNF cytotoxicity were suppressed by TGF-β1 (30--50%) (Fig. [7](#F7){ref-type="fig"}). However, TGF-β1 blocked TNF cytotoxicity of the GFP-expressing control cells to a greater extent (50--70%) (Fig. [7](#F7){ref-type="fig"}).

![TGF-β1 suppresses Hyal-1 and Hyal-2-increased TNF cytotoxicity in L929 cells. L929 cells stably expressing GFP, GFP-Hyal-1 or GFP-Hyal-2 were cultured in 96-well plates overnight, treated with TGF-β1 (2 ng/ml) for 2 hr, and then cotreated with TNF-α (5--40 ng/ml) for 24 hr. TGF-β1 inhibited TNF-α killing of L929 cells expressing GFP, and GFP-Hyal-1 or GFP-Hyal-2 (mean ± β1 inhibited TNF-α killing of the control GFP L929 cells to a greater extent (50--70%) than the GFP-Hyal-1 or GFP-Hyal-2 cells (30--50%).](1471-2121-3-8-7){#F7}

Discussion
==========

In this study, the novel function of the lysosomal hyaluronidases Hyal-1 and Hyal-2 in increasing cellular sensitivity to TNF cytotoxicity is reported. TGF-β1 counteracted Hyal-1- and Hyal-2-increased TNF killing of L929 fibroblasts. TGF-β1 increased L929 growth, while Hyal-1 and Hyal-2 blocked this enhancement of cell growth. These observations are consistent with our previous finding that exogenous bovine testicular hyaluronidase PH-20 enhances TNF cytotoxicity in various cancer cells, and that TGF-β1 blocks the increased TNF cytotoxicity \[[@B8],[@B15]\]. As summarized in the Figure [8](#F8){ref-type="fig"}, the underlying mechanisms whereby Hyal-2 induces TNF cytotoxicity appear to be associated with 1) Hyal-2 induction of the proapoptotic WOX1, 2) Hyal-2 migration to the mitochondria during apoptosis, and 3) the prolonged period of IκBα degradation and NF-κB activation in TNF-treated Hyal-2-expressing cells.

![A summarized scheme for Hyal-2-induced TNF susceptibility in L929 cells. Upregulation of proapoptotic WOX1 but not p53 was observed in the Hyal-2-expressing cells. Unlike PH-20, Hyal-2 could not induce constitutive JNK activation. TNF cytotoxicity was enhanced in these Hyal-2-expressing cells. The enhancement of TNF function is likely due to 1) the induced proapoptotic WOX1, 2) the likely translocation of Hyal-2 to the mitochondria in response to TNF, and 3) the prolonged period of IκBα degradation and NF-κB activation in as mediated by TNF.](1471-2121-3-8-8){#F8}

Upregulation of p53 and WOX1 contributes, in part, to the PH-20-enhanced cytotoxic function of TNF \[[@B9],[@B10],[@B23]\]. WOX1 is a p53-binding protein. Ectopic expression of both p53 and WOX1 cDNA constructs results in colocalization of these proteins in the mitochondria \[[@B23]\]. Increased synthesis and complex formation between p53 and WOX1 in the cytoplasm are observed during apoptosis \[[@B23]\]. Both p53 and WOX1 mediate apoptosis synergistically. Overexpressed WOX1 alone mediates apoptosis independently of p53 \[[@B10]\]. However, p53 apoptosis requires the participation of WOX1. Blocking WOX1 expression with antisense mRNA abolishes p53-mediated cell death, suggesting that WOX1 is a potential partner of p53 in apoptosis \[[@B10],[@B23]\].

Both Hyal-1 and Hyal-2 induced WOX1 expression when transiently expressed in L929 cells. However, only Hyal-2 induced WOX1 expression following stable transfection of L929 cells. This correlates with the greater extent of Hyal-2 enhancement of TNF killing compared to Hyal-1. WOX1 expression was induced by TNF in the Hyal-1 stable transfectant. In transient transfection experiments, Hyal-2 enhanced WOX1-mediated L929 cell death, while Hyal-1 had no effect. Both Hyal-1 and Hyal-2 alone failed to induce cell death. Thus, induction of WOX1 expression contributes, in part, to Hyal-1- and Hyal-2-increased TNF sensitivity in L929 cells.

TNF rapidly induces phosphorylation of IκBα, which normally peaks at 5--10 min, followed by proteasome-dependent degradation (10--20 min) \[[@B10],[@B24]\]. IκBα degradation and NF-κB activation occurred, as determined 20 min after exposure of control and Hyal-1 cells to TNF. However, there was an extended period of TNF-mediated IκBα degradation and NF-κB activation in Hyal-2 cells, ranging from 20--60 min. It is unclear whether this extend ed period of IκBα degradation and NF-κB activation directly contributes to the ability of Hyal-2 to enhance TNF-mediated apoptosis. IκBα is a physiological inhibitor of NF-κB activation; IκBα complexes with NF-κB and prevents its translocation to the nucleus \[[@B10],[@B24]\]. NF-κB activation is believed to induce antiapoptotic proteins to block cell death \[[@B8]\]. However, in several studies activated NF-κB cannot protect cells from apoptosis \[[@B8]\]. Also, NF-κB appears to be essential in the p53-mediated apoptosis in tested cell lines \[[@B25]\].

The mitochondrion is a reservoir of proapoptotic proteins and plays a key role in apoptosis \[\[[@B26],[@B27]\]; reviews\]. One of the pathways that leads to the release of proapoptotic proteins in the mitochondria has been determined. For example, the TNF/TNF receptor-signaling complex activates caspase 8, which leads to the activation and cleavage of Bid, a proapoptotic protein of the Bcl-2 family. Activated or truncated Bid (tBid) in turn activates Bak and Bax. Bak migrates to the mitochondrial outer membrane where it generates transmembrane pores; Bax induces the opening of the mitochondrial permeability transition pores. Cytochrome c is then released from the mitochondria into the cytosol, where it interacts with downstream proapoptotic proteins such as Apaf-1 and caspase 9. Activation of these proteins further activates nucleases, such as caspase-activated DNase (CAD), which cause DNA fragmentation.

During staurosporine-mediated apoptosis, a portion of the lysosomal Hyal-2 migrates to the mitochondria. Whether Hyal-2 causes mitochondrial membrane damage is unknown. Presumably, Hyal-2 interacts with a specific protein target on the mitochondrial surface that changes the membrane permeability. This notion is supported by our recent screening of Hyal-1 and Hyal-2 interacting proteins in yeast two-hybrid interactions (Chang *et al*, unpublished)

The functional property of Hyal-2 in degrading hyaluronic acid is controversial. Hyal-2 is found in the lysosome and is active under conditions of low pH \[[@B20]\]. Hyal-2 is also found on the cell surface as a glycosylphosphatidylinositol (GPI)-anchored receptor for jaagsiekte sheep retrovirus \[[@B21]\]. Nonetheless, the enzymatic activity of Hyal-2 has not been detected \[[@B21]\]. Whether the increased TNF cytotoxicity in L929 cells caused by Hyal-1 and Hyal-2 is associated with their enzymatic activities is being studied in this laboratory.

The TGF-β1 inhibition of Hyal-1 and Hyal-2 effects could be abolished by cycloheximide or actinomycin D (data not shown). That is, inhibition of protein synthesis or gene transcription reduced the inhibitory effect of TGF-β1 in the prevention of Hyal-1- or Hyal-2-increased TNF cytotoxicity. Whether TGF-β1-induced antiapoptotic proteins such as TIAF1 and TIF2 inhibit the function of Hyal-1 and Hyal-2 remains to be determined.

Conclusions
===========

Like testicular PH-20, lysosomal Hyal-1 and Hyal-2 enhanced TNF susceptibility in L929 cells. TGF-β1 suppressed the induced TNF sensitivity. Furthermore, TGF-β1-mediated L929 cell proliferation was blocked by Hyal-1 and Hyal-2. These data indicate that there is a functional antagonism between TGF-β1 and Hyal-1 or Hyal-2. Hyal-2-increased TNF susceptibility in L929 cells appears to be associated with 1) Hyal-2 upregulation of the proapoptotic WOX1, 2) Hyal-2 migration to the mitochondria during apoptotic cell death, and 3) the prolonged IκBα degradation and NF-κB activation in Hyal-2-expressing cells stimulated with TNF.

Materials and Methods
=====================

cDNA constructs, transient expression and stable transfectants
--------------------------------------------------------------

The full-length murine hyaluronidase *Hyal1* and *Hyal2* cDNAs were found in the EST database (Genbank accession AA688635 and BF139787, respectively) and purchased from Incyte Genomics (St. Louis, MO). The determined DNA sequences have been deposited in the GenBank (*Hyal1*, AF422176; *Hyal2*, AF422177). The coding regions were tagged with a *C*-terminal green fluorescent protein (GFP) sequence by subcloning into the *EcoR*1 site of the pEGFP-N1 vector (Clontech, Palo Alto, CA). Murine L929 cells were transfected with the constructed Hyal-1-pEGFP-N1 or Hyal-2-pEGFP-N1 vector, or a control pEGFP-N1 vector by electroporation (40 μg DNA/ 5 × 10^6^ cells; 250 volt and 960 Fd), as previously described \[[@B10]\]. The cells were cultured in the continuous presence of G418 (200 g/ml) for approximately 2 weeks, and the resulting total cell colonies were harvested. The presence of GFP-Hyal-1 and GFP-Hyal-2 proteins in these stable transfectants was examined by Western blotting using anti-GFP antibodies (Clontech). Where indicated, COS7 fibroblasts were transfected with the above constructs using a liposome-based reagent FuGene 6 (Roche, Indianapolis, IN) or GeneFector (Venn Nova, Pompano Beach, FL). Protein expression was visualized by fluorescence microscopy. Also, the cells were counterstained with LysoTracker or Mitotracker Red (Molecular Probes, Eugene, OR) to determine whether the expressed protein was located in the lysosome or mitochondria.

Cell lines and chemicals
------------------------

Murine L929 fibroblasts and monkey kidney COS7 fibroblasts were cultured as previously described \[[@B10]\]. Staurosporine, daunorubicin, actinomycin D, camptothecin and DAPI were from Calbiochem (San Diego, CA), Sigma (St. Louis, MO) and Biomol (Plymouth Meeting, PA). Purified human platelet TGF-β1 was from R&D (Minneapolis, MN). Antisera against WOX1 were generated in rabbits against a synthetic peptide \[[@B10]\]. Antibodies against p53, IκBα, NF-κB p65 and α-tubulin were from Santa Cruz Biotechnology (Santa Cruz, CA) and Accurate Chemicals (Accurate Chemical, Westbury, NY).

TNF cytotoxicity assay and Western blotting
-------------------------------------------

The above established cell lines were treated with TNF-α (2.5--20 ng/ml; Pepro-Tech, Rocky Hill, NJ) for 16--24 hr. The extent of cell death was determined by crystal violet staining \[[@B10]\]. Where indicated, the cells were also exposed to staurosporine, actinomycin D, daunorubicin, and camptothecin for 16--24 hr, before determining the extent of cell death. To determine whether TGF-β1 blocked Hyal-1 and Hyal-2-increased TNF killing, the cells were pretreated with TGF-β1 for 2 hr and then exposed to TNF-α in the continued presence of TGF-β1 for 16--24 hr. The extent of cell death was then determined. Where indicated, GFP-Hyal-1, GFP-Hyal-2 and GFP stable transfectants were treated with TNF (50 ng/ml) for 0, 20, 40, 60 and 120 minutes. The cells were extracted using NE-PER Nuclear and Cytoplasmic Extraction Reagents (Pierce, Rockford, IL) to separate the cytoplasmic and nuclear fractions. These preparations were subjected to SDS-PAGE and Western blotting using specific antibodies against IκBα, WOX1 and NF-κB.

Gene transfection and fluorescent microscopy
--------------------------------------------

COS7 cells were transfected with the GFP-Hyal-2 vector using the liposome-based FuGene 6 or GeneFector, cultured for 24--48 hr, and treated with staurosporine (1 M) for 0, 1, 2 and 4 hr. Mitochondria were stained with the membrane potential sensitive Mitotracker Red. Nuclei were stained by DAPI. The cells were then examined by fluorescence microscopy.

Acknowledgment
==============

Research was supported by the American Heart Association and the Guthrie Foundation for Education and Research. I thank Ms. Lori Schultz and Mr. John Heath for technical assistance, and Drs. Robert S. Aronstam, Margaret P. Quinlan and Victor Ruiz-Velasco for critically reviewing the manuscript.