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OPEN

Understanding of cell death
induced by the constituentsof Taxus yunnanensis wood
Yukihiro Akao1*, Riyako Terazawa1, Nobuhiko Sugito1, Kazuki Heishima1, Kohei Morikawa1,
Yuko Ito2, Ryoko Narui3, Reo Hamaguchi3 & Takahiro Nobukawa4
The ethanol extract from the wood of Taxus Yunnanensis (TY) induced apoptosis in all cancer cell lines
tested, which was mainly due to activation of an extrinsic pathway in human colon cancer DLD-1 cells.
The extrinsic pathway was activated by the upregulation of the expression levels of Fas and TRAIL/
DR5, which led to the activation of caspase-8. Of note, the machinery of this increase in expression
was promoted by the upregulation of MIR32a expression, which silenced MIR34a-targeting E2F3transcription factor. Furthermore, ectopic expression of MIR32a or siR-E2F3 silencing E2F3 increased
Fas and TRAIL/DR5 expression. Thus, the extract activated the extrinsic pathway through the MIR34a/
E2F3 axis, resulting in the autocrine and paracrine release of TRAIL, and upregulated expressionof death receptors Fas and DR5 in the treated DLD-1 cells, which were functionally validated by
Fas immunocytochemistry, and using anti-Fas and anti-TRAIL antibodies, respectively. In vivo, TY
showed significant anti-tumor effects on xenografted and syngeneic model mice. The extract may
also aid in chemoprevention by selectively making marked tumor cells susceptible to the tumor
immunosurveillance system.
The extract of Taxus yunnanensis wood has been used in traditional Chinese medicine by people in Yunnan
Province for treatment of the kidneys, diabetic ailments and other diseases. Currently, around 23,000 people
in Japan are taking it for healthcare. Chemical studies of the constituents and their functions, such as antirheumatic effects, have been ­reported1–6. On the other hand, paclitaxel is a potent anticancer drug that was
initially isolated from the bark of Taxus brevifolia1. Since the discovery of paclitaxel in 1­ 9711, much effort has
been devoted to isolate new taxane diterpenes with similar anticancer activity. As a result, more than 350 taxane
diterpenoids have been found from the bark of Taxus p­ lants2–4. The activity of most diterpenoids from bark ismarkedly high and toxic to healthy c­ ells5. However, the wood extract exerted minor effects on normal cells, but
it induced apoptosis mostly in cancer ­cells5. This difference in anti-proliferative activity is due to differences in
the constituents between the bark and wood. Although the pathways of cell growth and apoptosis have been
extensively investigated, it is still unclear how growth inhibition and apoptosis are regulated. Cancer cells enter
the cell cycle and progress by being stimulated by certain growth factors. On the other hand, apoptosis can beinitiated through one of two pathways: intrinsic pathway and extrinsic pathway. The intrinsic signaling pathways that initiate apoptosis through non-receptor-mediated stimuli that produce intracellular signals and are
mitochondrial-initiated events. Extrinsic pathways involve two pathways, death-receptor or non-death-receptor
mediated signal. In death-receptor mediated apoptosis of extrinsic pathway, two types of the pathways have been
defined: to be independent of mitochondria, called Type I and mitochondria dependent, called Type II. In thepresent study, we demonstrated that the apoptosis induced by the wood extract is mainly through an extrinsic
pathway induced by the greater upregulation of MIR34a silencing E2F2 (MIR34a/E2F axis). It has been reported
that phytochemicals affect epigenetic modifications, such as DNA methylation, and histone modifications inaddition to the regulation of the expression of non-coding microRNAs (miRNAs) for the prevention of c­ ancer6–8.
MiRNAs control numerous biological processes such as cell proliferation, apoptosis, and cell differentiation.
Due to their significant roles in cell physiology, alterations in expression levels are directly related to cancer
progression. Many phytochemicals have promise in regulating DNA methylation and histone modification in­carcinogenesis6–8, suggesting the use of dietary-based phytochemicals as potent and effective chemopreventive
medicines. In this study, the constituents of the extract upregulated MIR34a, which silenced the transcription
1

United Graduate School of Drug Discovery and Medical Information Sciences, Gifu University, 1‑1 Yanagido, Gifu,
Japan. 2Department of General and Gastroenterological Surgery, Osaka Medical and Pharmaceutical University,
Takatsuki, Japan. 3Karasuma Wada Clinic, Nakagyo‑ku, Kyoto 604‑0845, Japan. 4Kotosugi Co., Ltd, Sagamihara,
Japan. *email: yakao@gifu-u.ac.jp
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factor E2F3. In addition, death factors/receptors, such as FasL/Fas and TRAIL/DR5, were negatively regulatedby E2F3 after treatment in human colon cancer DLD-1 cells. Consequently, the extract induced apoptosis via
the activation of caspase-8 only in cancer cells through an extrinsic pathway by increasing the expression of suchdeath factors/receptors. This machinery is of great interest from the viewpoint of tumor immunology and cancer
prevention because of the minor effects on healthy cells.

Materials and methods

Preparation for extract of Taxus yunnanensis. The active ingredients of Taxus yunnanensis wereextracted from dried woodchips in 60% ethanol for 2 days. After evaporating the solvent, the bulk extract wasweighed and diluted in DMSO. The extract (TY) was stored at − 30 °C until use.
Cell lines and culture. DLD-1, DU145, K562, RPMI8226, and the other cells used in this study were cultured in RPMI1640 (Wako, Osaka, Japan) supplemented with 10% FBS (Sigma-Aldrich, MO, USA). H9c2 cells
were cultured in DMEM (Wako) supplemented with 10% FBS (Sigma-Aldrich). ASF 4-1 cells were cultured
in EMEM (Wako) containing 10% FBS (Sigma-Aldrich). All cell lines used were obtained from the JapaneseCancer Research Resources Bank except for DU145, which was obtained from the Riken Bioresource ResearchCenter. Cell lines used were confirmed as being negative for mycoplasma tMycoAlert, Lonza, Basel, Switzerland). All cells were cultured in a humidified incubator with 5% C
­ O2 at 37 °C.
Analysis of TY‑induced cell death. For cytotoxicity analysis, cells were seeded on a 6-well plate at a concentration of 0.5 × ­105 cells/mL. The cells were treated with TY (0, 1, 3, or 5 µg/mL). After a 48- or 72-h incubation, viable cells were counted using the trypan blue exclusion test. For morphological apoptotic analysis, theTY-treated and DMSO-treated cells were stained for 30 min with 5 μg/mL of Hoechst 33342 (Sigma-Aldrich)at 37 °C. After washing with PBS, nuclear morphology was imaged using a fluorescence microscope (Olympus,
Shinjuku, Tokyo, Japan). The apoptotic cell percentage was calculated by counting cells with fragmented nuclei
among 500 cells. For functional inhibitory assessment of apoptosis, chemical inhibitors of apoptosis (Z-VADFMK, Cat. 4800-520, MBL, Aichi, Japan; Z-IETD-FMK, Cat. 4805-510, MBL; Z-LEHD-FMK, Cat. 4810-510,
MBL) were used. For apoptotic analysis, DLD-1 cells were seeded at a concentration of 0.5 × ­105 cells/mL andincubated for 3 h. Subsequently, the cells were treated with certain concentrations of Z-VAD-FMK, Z-IETDFMK or Z-LEHD-FMK overnight. The cells were then treated with TY at 5 µg/mL for 72 h, followed by viable
cell count using the trypan blue exclusion test. For further functional inhibitory assessment of apoptosis, antiFas activating antibody (CH11: Sigma-Aldrich, Merck)9,10was used for synergistic activation of extrinsic apoptosis signaling by co-treatment with TY. Anti-TRAIL blocking antibody (2E5: Enzo Life Science, Cosmo Bio,
Japan)11 was used to inhibit extrinsic apoptosis signaling by co-treatment with TY.
Cell‑cycle analysis. Cell-cycle progression was analyzed by quantification of cellular DNA content using

the propidium iodide (PI) staining method (Tali Cell Cycle Kit, Thermo Fisher Scientific, MA, USA). Briefly,
DLD-1 cells treated with TY (5 or 10 µg/mL) or DMSO were washed twice with PBS and centrifuged at 500 × g
for 5 min. These cells were fixed with ice-cold 70% ethanol. After overnight incubation at − 20 °C, the cells were
centrifuged at 1000×g for 5 min at 4 °C. After a wash with 1 mL of PBS, the cells were stained with 200 μL of the
Tali Cell Cycle Solution containing PI for 30 min at room temperature in the dark. Subsequently, 25 μL of stained
cells was applied onto Tali Cellular Analysis Slides and analyzed with the Tali Image-Based Cytometer (Thermo
Fisher Scientific), and 20 fields per sample were captured with the function of the Tali Cell Cycle Assay. The dataobtained were analyzed in GraphPad Prism 8 (version 8.4.0, GraphPad Software, CA, USA).

Immunoblot.

To prepare cell lysate, DLD-1 cells treated with TY at 5 µg/mL for 24, 48, and 72 h were lysedin ice-cold RIPA buffer. In the case of tumor samples, the tumors were sonicated in 1% SDS buffer (Wako) andcentrifuged at 13,000×g for 20 min. The protein concentrations of the supernatants were measured using the
DC Protein Assay Kit (Bio-Rad, CA, USA). Protein samples (1–5 µg/lane) were subjected to sodium dodecylsulphate-polyacrylamide gel electrophoresis (SDS-PAGE). The proteins in the gel were transferred to 0.45-µm
polyvinylidene fluoride membranes (Immobilon-P Membrane, EMD Millipore, MA, USA)12. The membranes
were blocked with PVDF Blocking Reagent for Can Get Signal (TOYOBO, Osaka, Japan) for 1 h. Subsequently,
the membranes were incubated overnight at 4 °C with primary antibodies at a 1:1000 dilution. The membranes
were then washed 3 times with PBS containing 0.1% Tween 20, incubated further with HRP-conjugated sheep
anti-mouse or donkey anti-rabbit IgG antibody (Cell Signaling Technology [CST], Inc., Danvers, MA, USA)at room temperature, and then washed 3 times with PBS containing 0.1% Tween 20. The immunoblots werevisualized using the Luminata Forte Western HRP substrate (EMD Millipore)12. Densitometry of the immunoblots was performed using Image J (version 2.0.0-rc-69/1.52p, NIH). Primary antibodies used were as follows:
anti-FAS (#8023), DR5 (#8074), TRAIL (#3219), cyclin D1 (#55506), cleaved caspase-8 (#9496), PARP (#9542),
FADD (#2782), and phosphate-FADD (#2781; CST); anti-FAS ligand (#JM-3330-100) and caspase-8 (#M032-3;
MEDICAL & BIOLOGICAL LABORATORIES CO., LTD., Tokyo, JP); anti-E2F3 (SC-56665; Santa Cruz Biotechnology, Santa Cruz, CA, USA); and anti-SIRT1 and anti-Ras antibodies (ab32441; Abcam, Cambridge, UK).
The growth-related anti-phospho-ERK1/2 (#4370), anti-ERK1/2 (#4695), anti-phospho-AKT (Ser473) (#4060),
and anti-AKT (#9272) antibodies were obtained from CST. The loading protein amount was assessed with ananti-β-actin mouse monoclonal antibody (clone AC-74, Cat. #A5316, Sigma-Aldrich). Anti-rabbit IgG (#7074)
and anti-rabbit IgG, HRP-linked antibody (#7076; CST) were used as secondary antibodies.

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qRT‑PCR. Total RNA was extracted from DLD-1 cells treated for 24 or 48 h with TY at 5 µg/mL. In brief,

total RNA was purified using NucleoSpin miRNA (MACHEREY–NAGEL, Düren, Deutschland). The RNA concentration and integrity were assessed by UV spectrophotometry and Agilent 2100 Bioanalyzer (Agilent, CA,USA), respectively.
To detect mRNA, cDNA was reverse transcribed from 0.5 µg of the total RNA using the PrimeScript RT
Reagent Kit (Takara, Shiga, Japan)12. The cDNA was amplified with specific primers for FAS (Forward, 5’-GGA​
CCC​TCC​TAC​CTC​TGG​TT-3’; Reverse, 5’-ACC​TGG​AGG​ACA​GGG​CTT​AT-3’), TRAIL (Forward, 5’-ATC​ATG​
GCT​ATG​ATG​GAG​GT-3’; Reverse, 5’-CTG​T TC​ATA​CTC​TCT​TCG​TC-3’) and ACTB (Forward, 5’- GAT​TCC​
TAT​GTG​GGC​GAC​GA-3’; Reverse, 5’- AGG​TCT​CAA​ACA​TGA​TCT​GGGT-3’) by using Universal SYBR SelectMaster Mix (Applied Biosystems, Thermo Fisher Scientific, MA, USA). The signals were recorded using the
Cell line

Wood (ethanol extract)

Apoptosis

Human tumor cell line
SH-SY5Y (neuroblastoma)

0.98

+

DU-145 (prostate carcinoma)

1.92

+

PANC-1 (pancreatic carcinoma)

0.81

+

T24 (urinary bladder carcinoma)

2.62

+

MDA-MB-231 (breast adenocarcinoma)

0.56

+

Rh30 (rhabdomyosarcoma)

0.42

+

K562 (myelogenous leukemia)

2.56

+

RPMI8226 (myeloma)

1.2+

DLD-1 (colorectal carcinoma)

3.81

+

DLD-1/5FU

3.46+

DLD-1/OXA

3.52+

Non-cancerous cell lines
ASF-4-1 (human fibroblasts)

20.94

–

H9c2 (rat cardiocytes)

50 <

–

TIG-3-20 (human fibroblasts)

2.89

–

HMEC (human mammary epithelial cells)

50 <

–

Table 1.  Antiproliferative activity (IC50 value in μg/mL) of ethanol extract of Taxus Yunnanensis wood against
proliferating cells.

TaKaRa Thermal Cycler Dice Real Time System II (Takara). Relative expression levels were quantified by the ΔCt
method and normalized to ACTB. To detect miRNA, 25 ng of total RNA was reverse transcribed to cDNA using
the TaqMan MicroRNA Reverse Transcription Kit (Applied Biosystems)12. Expression levels of MIR34a wereassessed by performing TaqMan MicroRNA Assays (Applied Biosystems). The expression levels of MIR34a werenormalized using the ΔCt method and internal control (RNU6B). Each measurement was performed in triplicate.

Transfection of DLD‑1 cells with miRNA or siRNA. DLD-1 cells were seeded in 6-well plates at aconcentration of 0.5 × ­105 to 1.0 × ­105/1 mL/well on the day before transfection. MIR34a mimic (Applied Biosystems) or/and the inhibitor of MIR34a (anti-miR-34a, 40–80 nM; Applied Biosystems) or siRNAs for E2F3
(siR-E2F3: 5–10 nM) were used for the transfection of the cells, which was achieved by cationic liposomes,i.e., Lipofectamine RNAiMAX (Invitrogen, Carlsberg) according to the manufacturer’s lipofection protocol. The
sequence of anti-miR-34a was 5′-UGG​CAG​UGU​CUU​AGC​UGG​UUGU-3′; and that of siRNA for E2F3, 5′-UAA​
CCU​UUG​AUU​CUC​UGA​AUC​CUC​G-3′ (siR-E2F3). We used nonspecific control Duplex VII (57% GC Content; Dharmacon Research, Inc., Lafayette, CO, USA) as a control. The effects of the introduction of the miRNA,
siRNA, or anti-MIR34a inhibitor into the cells were assayed at 48 or 72 h after transfection.
Assay for luciferase activity.

We constructed sensor vectors by joining the region with or without a possible binding site from the 3′-UTR of human E2F3 (No. 4271-4650) with a luciferase reporter pMIR-control vector (Ambion, Foster City, CA, USA) to examine the target sequence of MIR34a. To generate sensor vectors withfour mutations in the binding site of the 3′-UTR of human E2F3 (No. 4440-4462) for MIR34a, we mutated seedregions from CACTGC​CA to CAAGTG​CA (mt-E2F3, PrimeSTAR Mutagenesis Basal Kit; TaKaRa). The sensor
vector with these mutations was submitted to Life Science Research Center, Gifu University, for DNA sequencing. The cells were seeded in 12-well plates at a concentration of 0.5 × ­105/well the day before transfection. The
sensor vector (concentration, 0.5 μg/well) and 40 nM MIR34a or nonspecific control miRNA (Dharmacon) wasused for the co-transfection of the cells with Lipofectamine RNAiMAX (Invitrogen). The measurement of theactivities was described ­previously12.

Immunocytochemistry.

After DLD-1 cells were treated with TY (4, 5, or 6 µg/mL) or DMSO for 48 h, thecells were fixed with 4% formaldehyde for 15 min and then incubated in 1% BSA/10% normal goat serum/0.3 M

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glycine in 0.1% PBS-Tween for 1 h to permeabilize the cells and block non-specific protein–protein interactions.
The cells were then incubated overnight at 4 °C with Alexa Fluor 647 Anti-Fas antibody (ab204671, Abcam)
at a 1:50 dilution. Nuclear DNA was labelled with Hoechst 33342. Images were taken by confocal microscopy(Leica-Microsystems).

Transmission electron microscopy (TEM). After DLD-1 cells were treated with TY (5 µg/mL) or
DMSO for 72 h, they were washed with ice-cold PBS. The cells were then fixed with 2% paraformaldehyde and2.5% glutaraldehyde in 0.2 M PBS for 2 h. After washing with PBS and post-fixing in 2% osmium tetraoxide for
2 h, the cells were dehydrated with a 10% graded series of 30–100% ethanol and cleared with QY-1 (Nissin EM,
Tokyo, Japan). The cells were then embedded in Epon 812 resin (TAAB Laboratories Equipment, Reading, UK)and sectioned at 70-nm thickness. The sections were stained with uranyl acetate and lead citrate. Transmission
electron microscopic images were obtained with a Hitachi-7650 operating at 80 kV (Hitachi, Tokyo, Japan).
Animal experiments. All mouse experiments were approved by the Institutional Animal Care and Use

Committee of Gifu University and performed according to the NIH Guide for the Care and Use of Laboratory
Animals (National Academies Press, 2011). Our in vivo study was reported accordance with ARRIVE guidelines(https://​arriv​eguid​elines.​org).
Their care was in accordance with guidelines of Gifu University. All mice were maintained with ad libitum
food and water under the standard light–dark cycle. Tumor volume was measured using the following formula:V = π/6(L × W2), where L, length; W, width of the tumor.
For the heterotopic colon cancer xenograft mice model, DLD-1 cells (5 × ­106 cells/100 μL of PBS) were
subcutaneously injected into the right flank of BALB/c nu/nu mice (female, 4 weeks old, SLC; n = 5). After themean tumor volume reached approximately 100 m­ m3, the mice were randomly separated into 2 groups. Subsequently, the mice were intraperitoneally administered (once in 2 days for a total of 3 injections) TY (50 mg/kg) or vehicle (0.1% DMSO in PBS). Two days after the final administration, the mice were sacrificed for theassessment of tumor tissues. For the heterotopic syngeneic colon cancer mice model, colon 26 cells (2 × ­106 cells
in 100 μL of PBS) were subcutaneously inoculated into female BALB/c mice (6 weeks old, SLC, Japan). Afterthe tumor volumes reached an average of 80 m
­ m3 in size, the mice were randomly allocated to different groups
based on their tumor size. The mice were treated with TY at 50 mg/kg or DMSO following an intraperitoneal
daily administration protocol for 20 days. The mice were euthanized with an overdose of isoflurane (20 mL in
an animal chamber; Pfizer, NY, USA). GraphPad Prism 8 (version 8.4.0, GraphPad Software, CA, USA) and JMP(version 12.2, SAS Institute, NC, USA) were used for statistical analysis.

TUNEL staining. TUNEL staining was performed with a TUNEL assay kit (ab206386, abcam). Briefly, tissues for the TUNEL stain were fixed in 4% neutral buffered paraformaldehyde, embedded in paraffin, and sectioned at 5 μm. The slides were deparaffinized in lemosol and rehydrated by passage through graded alcohols.
For antigen retrieval, the sections were incubated with Proteinase K solution. The sections were then incubatedin 0.3% hydrogen peroxide in methanol for 20 min to inhibit endogenous peroxidase activity. Subsequently, thesamples were labeled with TdT enzyme and FITC. After 3 washes with TBS-T, the sections were blocked withnormal serum and incubated with anti-FITC antibody conjugated with HRP. After 3 washes with TBS-T, thesignals were visualized with DAB chromogen solution. The slides were counterstained with hematoxylin. Histopathological images were acquired using a microscope (BZ-X700, KEYENCE).
Statistics. Differences were statistically evaluated by one-way analysis of variance followed by the t test. A
P-value of less than 0.05 was considered significant.

Results

TY induced cell death. In order to examine the growth inhibitory effects of the ethanol extract of thewood of Taxus Yunnanensis (TY), we tested 9 human cancer cell lines which have mutated p53 and 4 normal
cell lines, as shown in Table 1. The activity was markedly high against the tumor cells; IC50 values ranged from
0.4 to 4 μg/mL, in contrast to the normal cells other than human fibroblast TIG-3–20 cells (Table 1). We also
tested 5-fluorouracil (5-FU) and oxaliplatin (OXA)-resistant DLD-1 ­cells13 (Table 1). TY exerted high growth
inhibitory activity against the 5-FU or OXA-resistant DLD-1 cells, similar to the original DLD-1 cells (Table 1).
Morphologically, all cancer cell lines had apoptotic findings, such as chromatin condensation and nuclear fragmentation, on Hoechst 33342 staining. In order to examine the mechanism of apoptosis, we focused on a humancolon cancer DLD-1 cell line among the cell lines because it exhibited typical apoptotic changes by Hoechst
33342 staining (Fig. 1A). Cell-cycle analysis revealed a major population of sub-G1 phase after treatment with
5 μg/mL for 48 h (Fig. 1B), which indicated a typical apoptotic feature. Biochemically, the apoptosis was induced
in a caspase-dependent manner, which was evaluated by Western blot analysis (Fig. 1C). The cleaved forms ofpoly (ADP-ribose) polymerase (PARP) that catalyzed by caspase-3 clearly emerged at 24 h after the treatment,along with the cleaved forms of caspase-8 and caspase-9, suggesting that TY induced apoptosis via both extrinsic
and intrinsic pathways. Survival signal pathways of PI3K/Akt and MAPK/Erk were relatively activated up to 48 h
after the treatment, but returned to normal at 72 h. The expression levels of KRAS and IκB were also unchanged
during 72 h after treatment. A transition from LC3B-1 to LC3B-II, a biochemical marker of autophagy, wasobserved (Fig. 1C). Electron microscopic study demonstrated typical apoptotic findings accompanied by theformation of several autophagosomes in the same cell (Fig. 1D). Thus, TY induced apoptosis by activating both
apoptosis pathways and altering autophagic cell death in DLD-1 cells. Next, we performed cell biological examination using caspase inhibitors. As expected, significant apoptosis inhibitory effects of Z-VAD-FMK, a panScientific Reports |Vol:.(1234567890)(2022) 12:6282 |https://doi.org/10.1038/s41598-022-09655-2

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Figure 1.  Growth inhibition by TY (the ethanol extract from Taxus Yunnanensis wood) in DLD-1 cells. Effects of TY on the cell
growth in the colon cancer cell line DLD-1. (A) The cell viability was estimated at 72 h after treatment. Data were obtained from atleast three independent experiments. Data for the representative concentrations are shown. The cell viability of the control (DMSO
alone) is indicated as 100%. The growth inhibitory activity ­(IC50) of TY is indicated in Table 1. *P < .05 is significant. Apoptotic cell
death is induced by TY. Apoptotic cells (white arrows) at 48 h after treatment were shown by Hoechst 33,342 staining. (B) Cell cycle
analysis in TY-treated cells. Cell-count percentage of G0/G1 (2 N), S, and G2/M (4 N) phases for DLD-1 cells treated with TY (5 or
10 µg/mL) or DMSO. ****P < 0.0001, 1-way ANOVA with Dunnett’s post hoc test. Data are presented as the mean ± SD (n = 3). NS, notsignificant. (C) Time-dependent activation of the apoptotic signaling cascade in comparison with the growth signal cascade after thetreatment with 5 μg/mL of TY, which was estimated by Western blot analysis. Arrows indicates cleaved forms of caspases and LC3B I
and II. (D) Electron microscopic findings in TY-treated DLD-1 cells. The cells were treated with 3 μg/mL of TY for 72 h.

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Figure 1.  (continued)
caspase inhibitor, were noted based on the viable cell rate and Hoechst 33342 nuclear staining (Fig. 2A), similar
to the case of the capase-8 inhibitor, Z-IETD-FMK (Fig. 2B). Furthermore, we examined the inhibitory effect of
caspase-9 inhibitor, Z-LEHD-FMK on TY-induced apoptosis to verify the signaling via mitochondria. As shownin Fig. 2C, a significant inhibition of the apoptosis was found.
Based on the experimental results regarding apoptosis, we next explored which death receptor(s) functioned in the extrinsic apoptosis signaling induced by the wood extract of Taxus Yunnanensis. We examinedthe expression of Fas/FasL and TRAIL/DR5 after treatment. As shown in Fig. 3A, the expression levels of FAS
mRNA and protein in the treated cells significantly increased at 48 h and the increased protein expression
level remained up to 72 h, which may lead to the upregulation of p-FADD (Fig. 3A). Thus, it is confirmed that
activation of caspase-8 from death-receptors play a pivotal role in this apoptosis. Of note, the expression levelsof TRAIL and DR5 markedly increased at the same time. To examine which transcription factor regulates theexpression of FAS, TRAIL, and DR5, we surveyed the changes in the expression levels of certain transcription
factors and tumor suppressor miRNAs by Western blot and qRT-PCR, respectively. As a result, we found markedly increased expression levels of MIR34a at 48 h after TY treatment compared with the control by qRT-PCR(Fig. 3B). Accordingly, to assess the role of MIR34a in the growth suppression along with apoptosis, we induced
its ectopic expression in DLD-1 cells, which resulted in significant growth inhibition with apoptotic characteristics, such as chromatin condensation and fragmentation, on Hoechst 33342 staining (Fig. 3C). The miRNAdatabase (miRbase) suggested that MIR34a, one of the major tumor suppressor miRNAs, silences E2F3, SIRT1,
and CyclinD1. We thus examined the protein expression of the transcription factor E2F3, SIRT1, and CyclinD1
after treatment. The expression of E2F3, SIRT1, and CyclinD1 was significantly downregulated from 24 h up to
48 h (Fig. 3D). Next, the silencing of E2F3 by MIR32a (20 nM) transfection with the activation of caspase-8 waspartly canceled by co-transfection with anti-MIR32a in a dose-dependent manner (Fig. 3E). Thus, the MIR34a/
E2F3 axis was established in apoptosis after the treatment with TY. Furthermore, we confirmed the increased
expression levels of the death receptors DR5 and TRAIL in the MIR34a-trasfected cells at 72 h (Fig. 3F). We
thus examined whether TRAIL/DR5 are the target genes of E2F3. To assess transcriptional control of DR5 and
TRAIL by E2F3, we silenced E2F3 by siR-E2F3. As expected, DR5 and TRAIL were both upregulated according
to Western blot analysis (Fig. 3F). Therefore, E2F3 may control the inhibitory machinery of the expression of
Fas and TRAIL/DR5. The luciferase reporter assay for targeting E2F3 by MIR34a was performed and the activity was reduced by the ectopic expression of MIR34a, but partly canceled in the case of a mutated binding site
(Fig. 4A–C). As for FAS, a dose-dependent increase in the expression level was observed in the cytoplasm byimmunocytochemical study (Fig. 5A).

Functional assays of the increased FAS and TRAIL/DR5 induced by TY using CH‑11 and2E5. We next examined whether the increased expression levels of FAS and TRAIL/DR5 by TY treatment

contribute to this apoptosis. Expectedly, the activating antibody for FAS (CH-11)15,16 synergistically induced
apoptosis based on the viable cell rate and Hoechst 33346 nuclear staining after combined treatment with TY(Fig. 5B). To further confirm whether TY induces TRAIL/DR5-driven apoptosis in the treated DLD-1 cells, we
examined whether anti-TRAIL antibody (2E5)11 can block the apoptosis elicited by TY. As shown in Fig. 5C, theantibody efficiently blocked the activation of caspase-8 through TRAIL/DR5 binding, similar to the case of the
caspase-8 inhibitor. Thus, the upregulated Fas, TRAIL, and DR5 via MIR34a/E2F3 axis function in TY-induced
apoptosis (Fig. 6).

Anti‑tumor effects of TY on mouse models. In order to examine the anti-tumor effects of TY in in vivo
mouse models, we produced 2 different mouse models, DLD-1 cell xenograft and syngenic tumor-bearing mice.
In DLD-1 cell xenograft mice, the treatment was performed twice a week, for 4 times in total. Significant antitumor effects were found in the treated group compared with the control group (Fig. 7A). However, no apoptosis
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Figure 2.  Effects of caspase inhibitors on apoptosis induced by TY (5 μg/mL) in DLD-1 cells. (A) Inhibition of
TY-induced apoptosis by combined treatment with pan-caspase inhibitor Z-VAD-FMK at 72 h after treatment
with TY. Hoechst 33348 staining of treated cells (upper figure). Rescue of the viable cell rate in the TY-treatedcells by co-treatment with or without the inhibitor (lower figure). (B) Rescue of the viable cell rate in the
TY-treated cells by co-treatment with or without caspase-8 inhibitor Z-IETD-FMK at 72 h after the treatment
with TY. (C) Rescue of the viable cell rate in the TY-treated cells by co-treatment with or without caspase-9
inhibitor Z-LEHD -FMK at 72 h after the treatment with TY.

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Figure 3.  TY (5 μg/mL) induced the expression of the death factors/receptors Fas L/Fas and TRAIL/DR5. (A) The expression
levels of FAS mRNA at 48 h after TY treatment estimated by qRT-PCR (left figure). Time-dependent protein expressionof p-FADD and death factors/receptors Fas-L/Fas and TRAIL/DR5 were examined by Western blot analysis. (B) Timedependent expression of MIR34a after treatment with TY estimated by qRT-PCR. Expression levels of MIR34a are shownas the relative ratios with respect to the RNU6B expression levels. (C) Ectopic expression of MIR34a inhibited cell growthdose-dependently and induced apoptosis according to Hoechst 33348 staining at 72 h after transfection. The apoptotic cells areindicated by white arrows. (D) Expression of MIR34a-targeting E2F3, SIRT1, and CyclinD1 was downregulated by the TY. (E)Anti-MIR34a (Anti; 10 and 20 nM) partly canceled the activation of caspase-8 and PARP cleavage elicited by co-transfection
with MIR34a. (F) Silencing of E2F3 by siR-E2F3 or MIR32a increased in the expression levels of DR5, TRAIL, and Fas.
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Figure 3.  (continued)

Figure 4.  MIR34a binds to E2F3 mRNA in the Luciferase assay. (A–C) The ORF region of human E2F3 mRNAand the predicted binding site for MIR34a (the sequence region 4440–4462) were inserted into a pMIRREPORT Luciferase miRNA Expression Reporter Vector (wild). The mutant-type pMIR vector contained
a mutated seed sequence (from CTGC to AGTG) for MIR34a. Luciferase activities were measured afterco-transfection with control RNA or MIR34a (10 nM) and wild or mutant pMIR vector.

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Figure 5.  TY upregulated the expression of Fas that functions as a caspase-8-dependent apoptosis inducer.
(A) Immunocytochemistry for Fas in TY-treated cells. The expression levels of Fas in the cytoplasm increased
dose-dependently (4, 5, and 6 μg/mL). Nuclear Hoechst 33348 staining was also performed. (B) Fas-activating
anti-Fas antibody (CH-11) synergistically induced apoptotic cell death by co-treatment with TY, which wasestimated by the viable cell rate and Hoechst 33342 staining. (C) TRAIL-blocking anti-TRAIL antibody (2E5)
partly canceled apoptotic cell death induced by TY through suppressing the activation of caspase-8, which wasestimated by the viable cell rate and Western blot analysis of caspase-8.

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Figure 6.  Schematic of the mechanism of TY-induced apoptosis in DLD-1 cells.

in the syngenic graft tumors of treated mice was observed. In syngenic graft mice, Colon 26 cells were cutaneously inoculated into the back and the extract was intraperitoneally administered every day at a dose of 50 mg/
kg. As shown in Fig. 7B, the tumor growth in the treated group was suppressed compared with that in the control(DMSO) group. Protein samples from TY-treated tumors demonstrated the activation of caspase-3 (Fig. 7C).
Pathologically, HE staining of the tumor samples confirmed the coexistence of macrophages and apoptotic cellswith pyknotic nuclei in the interstitial region. TUNEL study revealed many positive cells in the area (Fig. 7D).

Discussion

We demonstrated that the natural components included in the wood of Taxus Yunnanensis induce apoptotic cell
death partly through activating an extrinsic pathway, in which death ligands and receptors were upregulated by
TY. TY induced apoptosis in tumor cells in an autocrine or paracrine manner via TRAIL or FasL from treatedtumor cells. This suggests that the increased expression levels of Fas and TRAIL/DR5 by TY make tumor cells
susceptible to attack by tumor-related immune cells such as cytotoxic T cells and NK cells. We also clarified themechanism of upregulation of cell death ligands/receptors through the MIR34a/E2F3 axis toward apoptosis.
TY markedly increased the expression level of MIR34a, which silenced E2F3 by 36–48 h after treatment. As
expected, decreased expression of E2F3 by siR-E2F3 upregulated Fas and TRAIL/DR5. Thus, E2F3 negatively
regulated their mRNA transcription. Furthermore, we noted increased expression of Fas in the cell cytoplasm
by immunocytochemistry 48 h after the treatment with TY (Fig. 5A), and anti-Fas activating antibody (CH11) synergistically promoted the growth inhibitory activity when combined with TY (Fig. 5B). On the other
hand, the antibody against TRAIL efficiently inhibited the apoptosis elicited by TY, which suggested the releaseof TRAIL from the DLD-1 cells treated with TY (Fig. 5C). The MIR34a/E2F3/death ligand-receptor cascade
induced by TY functions in the apoptosis. Thus, TY induced apoptosis through MIR34a/E2F3, leading to deathligand-receptor cascades.
miRNAs are a group of small non-coding RNAs functioning in target gene regulation at the transcriptional or
post-transcriptional level. Accordingly, miRNA can function as an oncogene or tumor suppressor by regulating
growth or death-associated genes in human cancer cells. MIR34a is a typical tumor suppressor miRNA, whosetranscription factor is p53, which is deleted in most cancers and DLD-1 c­ ells14. The current study suggested thatthese bi-directional circuits exist widely among miRNA/target transcription factor ­genes15–17. Reinforcing theinhibition of transcripts in specific target cells is one prominent biological function of miRNAs. Some miRNAs
silence E2F3 and several of them are also regulated by E2F3 through an auto-regulatory feedback l­oop18. We
revealed that MIR34a targets E2F3 by Western blot analysis and Luciferase assay. As the ectopic expressionof MIR34a and siRNA silencing of E2F3 both upregulated Fas and TRAIL/DR5 protein expression, miR34a/
E2F3/Fas and TRAIL/DR5 cascades may exist. Phytochemicals impact epigenetic modifications, such as DNA
methylation, and histone modifications, in addition to the regulation of non-coding miRNA expression for theprevention of ­cancer7,8. Due to their significant roles in cell physiology, expression level alterations are directly
related to cancer progression. In this study, the phytochemicals of TY increased the expression level of MIR34a,
which may be associated with demethylation of the promoter region of MIR34a because the promoter of MIR34a
is frequently hypermethylated in colon cancer ­cells19–21. Many phytochemicals have promise in altering DNA

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Figure 7.  Anti-tumor activity of TY in mouse models. (A)Tumor volume (raw and relative) and body weight in the experiments toassess the anti-proliferative effects of TY (50 mg/kg) in the DLD-1 xenograft mouse model (n = 5). Dots indicate the administration ofTY. *P < 0.05, two-tailed, unpaired Student’s t-test. Data are presented as the mean ± SEM. (B) Tumor volume, body weight, and grossimages of tumors to assess the anti-proliferative effects of TY (50 mg/kg) in the Colon 26 syngeneic mouse model (n = 8). **P < 0.01,
two-tailed, unpaired Student’s t-test. Data are presented as the mean ± SEM. TY was given by peritoneal injection every day for 20 days.
Dots indicate the administration of TY. The tumor samples were analyzed with immunoblots for two apoptosis markers (cleaved
caspase 3 and PARP), and the densitometric values are indicated along with the blots. (C) Representative images and positive cell counton TUNEL staining of the tumor tissue of mice shown in Fig. 7B. Data are presented as the mean ± SD. *P < 0.05, two-tailed, unpaired
Student’s t test.

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methylation and histone modifications in carcinogenesis, which may lead to the use of dietary-based phytochemicals as potent and effective chemopreventive medicines.
TY induced the autocrine and/or paracrine release of FasL/Fas and TRAIL/DR5 in treated DLD-1 cells. As for
TRAIL signaling, TRAIL-R2/DR5 was used for apoptosis in this case because the TRAIL antibody blocked theactivation of caspase-8. The induction of apoptosis can be classified into either intrinsic apoptosis, triggered by
p53 in response to cellular ­damages22, or extrinsic apoptosis induced upon death ligand binding to a death receptor. Conventional anticancer drugs induce intrinsic apoptosis through p53 in response to cellular d­ amage22,23.
However, most tumors have mutations in p53, leading to its inactivation and failure of chemotherapeutic agents.
In contrast, TRAIL-induced apoptosis remains possible in many tumors despite non-functional p53. Therefore,
the effects of TY on TRAIL signaling and sensitization may be useful for cancer prevention and therapy. Two
extrinsic pathways from death-receptors can be distinguished based on the requirement of cross-signaling mitochondrial apoptotic ­machinery24. In type I cells, DISC activation is sufficient to effectively induce the full caspasecascade. In type II cells, DISC activation causes caspases-9 and -3 activation by signaling pathways via mitochondria. In this case, the mitochondria membrane potential is not downregulated and activation of caspase-9
is clearly observed. In addition, caspase-9 inhibitor considerably blocked the apoptosis. It is reasonable that theextrinsic pathway of type II is the major machinery in this apoptosis. When tumor necrosis factor (TNF)-related
apoptosis-inducing ligand (TRAIL) was discovered in 1995, many studies were performed to demonstrate that it
selectively induces apoptosis in tumor cells in vivo25–27. At present, TRAIL-related therapy against human cancers
is under investigation. The use of TY with the ability to selectively kill tumor cells through the TRAIL-related
signaling pathway is preferable. On the other hand, as shown in Fig. 1C, the expression levels of Akt/p-Akt,
ERKs/p-ERKs and KRAS at 72 h after the TY treatment almost unchanged. Therefore, the survival signals fromgrowth factors such as epidermal growth factor (EGF) and fibroblast growth factor (FGF) would sustain during
activation of the extrinsic pathway. Thus, it seemed that TY does not affect the survival signal pathways. Thus,
our study suggested that the wood extract from TY selectively kills tumor cells by inducing apoptosis throughthe increased expression of cell-death ligands/receptors, which also make the marked tumor cells susceptible tothe immunosurveillance system. The similar findings were also observed in other colon cancer cell lines.
TY contains many diterpenoids, such as baccatin III, 10-deacetyltaxuyunnanine C, and taxuyunnanine E,
which may have anti-proliferative e­ ffects5. However, most are cytotoxic to healthy cells. Cytotoxic 10-deacetyltaxuyunnanine C induced the activation of caspase-8, but not the increased expression of cell-death factor/receptors. The anti-cancer effects of TY may be due to synergy between diterpenoids and other factors such as lignans.
Received: 5 December 2021; Accepted: 21 March 2022

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Acknowledgements

This study was financially supported by a KOTOSUGI grant.

Author contributions

Data curation: Y.A., R.T. Formal analysis: Y.A., Y.I., K.M., K.H., N.S., R.N., R.H. Funding acquisition: Y.A.
Supervision: Y.A., T.N. Writing-draft: Y.A., R.T.

Competing interests

The authors declare no competing interests.

Additional information

Correspondence and requests for materials should be addressed to Y.A.
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