Habib et al. BMC Molecular and Cell Biology
https://doi.org/10.1186/s12860-024-00501-z(2024) 25:5

BMC Molecular andCell Biology

Open Access

RESEARCH

Optimizing combination therapy in prostatecancer: mechanistic insights into the synergistic
effects of Paclitaxel and Sulforaphane-induced
apoptosis
Tito N. Habib1* , Mohammed O. Altonsy1,2, Salah A. Ghanem1, Mohamed S. Salama3 and Mai A. Hosny1

Abstract
Background Combination therapies in cancer treatment have demonstrated synergistic or additive outcomeswhile also reducing the development of drug resistance compared to monotherapy. This study explores the potentialof combining the chemotherapeutic agent Paclitaxel (PTX) with Sulforaphane (SFN), a natural compound primarilyfound in cruciferous vegetables, to enhance treatment efficacy in prostate cancer.
Methods Two prostate cancer cell lines, PC-3 and LNCaP, were treated with varying concentrations of PTX, SFN,
and their combination. Cell viability was assessed using the thiazolyl blue tetrazolium bromide (MTT) assay to determine the EC50 values. Western blot analysis was conducted to evaluate the expression of Bax, Bcl2, and Caspase-3
activation proteins in response to individual and combined treatments of PTX and SFN. Fluorescent microscopy
was employed to observe morphological changes indicative of apoptotic stress in cell nuclei. Flow cytometry analysis
was utilized to assess alterations in cell cycle phases, such as redistribution and arrest. Statistical analyses, including Student’s t-tests and one-way analysis of variance with Tukey’s correction, were performed to determine significant differences between mono- and combination treatments.
Results The impact of PTX, SFN, and their combination on cell viability reduction was evaluated in a dose-dependent
manner. The combined treatment enhanced PTX’s effects and decreased the EC50 values of both drugs compared
to individual treatments. PTX and SFN treatments differentially regulated the expression of Bax and Bcl2 proteinsin PC-3 and LNCaP cell lines, favoring apoptosis over cell survival. Our data indicated that combination therapy significantly increased Bax protein expression and the Bax/Bcl2 ratio compared to PTX or SFN alone. Flow cytometry analysis
revealed alterations in cell cycle phases, including S-phase arrest and an increased population of apoptotic cells.
Notably, the combination treatments did not have a discernible impact on necrotic cells. Signs of apoptotic cell death
were confirmed through Caspase-3 cleavage, and morphological changes in cell nuclei were assessed via western
blot and fluorescent microscopy.
Conclusion This combination therapy of PTX and SFN has the potential to improve prostate cancer treatment
by minimizing side effects while maintaining efficacy. Mechanistic investigations revealed that SFN enhances PTX
efficacy by promoting apoptosis, activating caspase-3, inducing nuclear morphology changes, modulating the cell
cycle, and altering Bax and Bcl2 protein expression. These findings offer valuable insights into the synergistic effects*Correspondence:
Tito N. Habib
titohabib99@science.sohag.edu.eg
Full list of author information is available at the end of the article© The Author(s) 2024. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which
permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to theoriginal author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images orother third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line
to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory
regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of thislicence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.

Habib et al. BMC Molecular and Cell Biology(2024) 25:5

Page 2 of 14

of PTX and SFN, supporting the optimization of combination therapy and providing efficient therapeutic strategies
in preclinical research.
Keywords Sulforaphane, Paclitaxel, Prostate Cancer, Cell cycle, Apoptosis, Combination therapy, Drug synergy

Background
Combination therapy is a recommended intervention inwhich the patient receives more than one therapy. Treatment regimens involving the administration of several
separate pills, each containing a specific drug, or single
pills containing different drugs are examples of combination therapy.
Previous studies have investigated the combinatory
effects of SFN and PTX in various cancer cell types,
including breast [1], ovarian [2], lung [3], and prostatecancer [4]. These studies have consistently shown thatthe combination treatment can synergistically inhibit cell
growth and induce apoptosis and that this effect is associated with an increase in ROS production and a decreasein Bcl2 expression. These findings suggest that SFN may
have the potential as an adjuvant therapy to improve theefficacy of PTX in treating various types of cancer.
Prostate cancer is the sixth leading cause of death inmen worldwide, with over one million men diagnosed
with prostate cancer and over three hundred thousand
deaths in 2012 alone. These numbers rank prostate cancer as the second most commonly diagnosed cancer inmen [5]. The prostate gland secretes alkaline fluid in themale reproductive system as part of semen, which functions as a pH buffer to protect the sperm [6]. Enlargementof the prostate gland is a common age-related symptom
known as benign prostatic hypertrophy (BPH), which,although noncancerous, can result in unpleasant clinicalsymptoms such as urination problems, infections, andkidney diseases [7]. The development of prostate cancer
is gradual and occurs over a prolonged period, making
early diagnosis challenging. The etiology of the disease
is widely heterogeneous, with genetics, ageing, obesity,and ethnicity identified as major risk factors [8]. Recently,
researchers have studied the role of dietary habits in theincidence of prostate cancer, highlighting high intakes ofdairy products, red meats, processed meats, and foods
rich in α-linolenic acid, and calcium as possible risk factors [9–11]. Such research has created a groundswell ofinterest in studying the influences of certain dietary components on human health and disease status.
Sulforaphane (SFN), a compound found naturally incruciferous vegetables, has potential therapeutic properties, including detoxification, antimicrobial, anti-inflammatory, and redox balancing [12]. SFN’s therapeutic andprotective properties may be attributed to the induction of the nuclear factor-erythroid-2-related factor 2(Nrf2) transcription factor, which regulates antioxidant
response elements, inflammation, non-enzyme antioxidants, and phase II detoxification enzymes [13]. SFN has
been shown to induce the expression of quinone reductase and glutathione transferases, phase II anticarcinogenic enzymes, in murine hepatoma cells [14–16]. SFN
has been focused on by researchers and utilized to treat
different types of cancers due to its promising therapeutic properties [17].
The role of SFN in human prostate cancer was previously studied by researchers who reported potent induction of phase II enzymes and initiation of reactive oxygen
species following SFN treatment [18, 19]. SFN has also
shown a protective effect against prostate cancer recurrence and significantly lowered the prostate-specific antigen (PSA) progression after radical prostatectomy [20].
Additionally, SFN decreased upregulated histone deacetylase (HDAC3) protein expression in transgenic adenocarcinoma of the mouse prostate [21].
In cancer treatment, combined therapy results in synergistic or additive outcomes and reduces the developmentof drug resistance in response to anticancer agents compared with monotherapy [22]. Paclitaxel (Taxol, PTX) isan anticancer drug that targets actively dividing cells byhalting their mitosis, arresting cell growth, and ultimately
initiating apoptotic cell death [23]. Here, we compare theoutcomes of combined and monotherapies of SFN and
PTX in prostate cancer cell lines (PC-3 and LNCaP). We
apply molecular biology techniques to measure apoptosis, cell cycle arrest, and the expression of Bax and Bcl2
proteins in response to the two different treatment strategies. Understanding the mechanism of drug synergy,
as opposed to simply knowing which drugs to combine,
enables further optimization of advantageous drug interactions and can provide efficient therapeutic strategies in preclinical research. Such research could lead tothe development of new biomarkers and guide therapy
choices, ultimately improving the treatment outcomesfor patients with prostate cancer.

Materials and methods
Cell lines and reagents

The prostate cancer adenocarcinoma cell line PC-3
(catalog no. CRL-1435; ATCC) and prostate carcinoma
cell line LNCaP (catalog no. CRL-1740; ATCC) wereobtained from ATCC, Egypt. The cells were cultured

Habib et al. BMC Molecular and Cell Biology(2024) 25:5

in RPMI-1640 complete growth medium (catalog no.
12633-012; Gibco) supplemented with 10% heat-inactivated fetal bovine serum (FBS) (catalog no. 098105;
Multicell). The seeded cells were incubated at 37 °C and
5% ­CO2 in a humidified incubator, and the medium waschanged every 48 h. Once the cells reached 80% confluency, they were dissociated using 0.25% trypsin (catalog
no. 15400-054; Gibco) and plated in 24-well cell culture
plates at a density of 70,000 cells per well for 24 h before
treatment.
Paclitaxel (PTX) (catalog no. T7402; Millipore Sigma)
was dissolved in dimethyl sulfoxide (DMSO) (catalog no.
d5879; Sigma-Aldrich) at a concentration of 50 mg/mL.
Sulforaphane (SFN) (catalog no. s4441; Millipore-Sigma)
was diluted in DMSO to a concentration of 5 mg/mL.
Different concentrations of PTX or SFN were freshly prepared in a complete culture medium before treatment.
Cell viability assay

To examine the effect of PTX and SFN on PC-3 cell viability, cells were grown in complete medium in 48-wellplates at a density of 35,000 cells per well for 24 h before
treatment. To investigate the effect of PTX in combination with SFN on PC-3 cell viability, we dissolved bothdrugs at equal concentrations starting at 100 ng/ml to2500 µg/ml. Thiazolyl blue tetrazolium bromide (MTT)(catalog no. m-5655; Sigma) was used for the cell viability
assay. MTT was dissolved in phosphate-buffered saline
at a concentration of 5 mg/mL. After treating the cellswith PTX, SFN, or PTX + SFN, MTT was added to eachwell of 24-well plates at a final concentration of 1 mg/
mL directly to the culture medium. The plates were thenincubated at 37 °C for three hours.
The culture medium was then removed, and the MTT
formazan crystals were dissolved in 500 µL of MTT solvent (4 mM HCL, catalog no. acs393; BDH, 0.1% Nonidet P40, catalog no. 74,385; Fluka, in isopropyl alcohol,
catalog no. un1219; Omnisolv) on a rocker in the dark for
15 min.
Then, 100 µL of the dissolved MTT crystals were transferred to each well in a 96-well plate and were read on a
SPECTRAmax PLUS384 Microplate spectrophotometer
set to a 590 nm wavelength. The absorbance values wereused to calculate the percentage of viable cells relative tothe untreated control cells.
Cell lysate preparation, total protein quantification,and Western blot analysis

After the treatments, the culture medium was removed,and cells were harvested in Radioimmunoprecipitation
assay buffer (RIPA) containing a 1x protease inhibitor
cocktail (catalog no. PI-78439c; Thermo Scientific).

Page 3 of 14

Total protein quantification was conducted using the
BioRad protein assay (catalog no. 500-0006; BioRad)following the manufacturer’s protocol.
Total protein was denatured by adding 2x Laemmli
buffer (SDS, 4%; β-mercaptoethanol, 10%; glycerol,
20%; bromophenol blue, 0.004%; Tris-HCl, 0.125 M)
in a 1:1 (v/v) ratio and boiled at 95 °C for 5 min. Then,
50 µg of protein per sample was loaded into 10% SDSPAGE. BLUelf pre-stained protein ladder (catalog no.
PM008-0500; Frogga Bio) was used as a molecularweight marker (5-245 kDa).
The separated protein bands were transferred to anitrocellulose membrane (catalog no. rpn203D; EG
Healthcare). The membranes were immune-probedwith rabbit polyclonal anti-caspase-3 (catalog no. AAP113E; Stressgen), rabbit monoclonal anti-Bax (catalog no. ab32503; Abcam), rabbit monoclonal anti-Bcl2(catalog no. ab32124; Abcam), and anti-glyceraldehyde3-phosphate dehydrogenase (anti-GAPDH) (catalog no.
4699–9555; Biogenesis).
To detect the immune-probed protein bands, we usedperoxidase-affiniPure goat anti-mouse IgG (catalog no.
115-035-003; Jackson ImmunoResearch) or peroxidaseconjugated goat anti-rabbit IgG (catalog no. 111-035003) as secondary antibodies. Band visualization anddensitometric analysis were carried out using Pierce
ECL Western Blotting Substrate (catalog no. PI-32,106;
Thermo Fisher Scientific), Chemi Doc XRS system, andImage Lab 6.0 software (BioRad).
Fluorescent microscopy and image analysis

Cells were grown on coverslips placed at the bottom ofeach well in 24-well plates at a density of 70,000 cells
per well for 24 h before treatment.
Following treatment, the cells were fixed in ice-coldmethanol (catalog no. a412; Fisher Chemicals) for
10 min at -20 °C. Methanol treatment permeabilized
the cell membrane and allowed 4’,6-diamidino-2-phenylindole (DAPI, catalog no. d21490; Molecular Probes)
to penetrate and stain the nuclear chromatin. DAPI was
prepared in phosphate-buffered saline (PBS) at a concentration of 300 nM and added to the cell monolayers for 5 min, followed by three washes (5 min each) in
PBS.
Cells were then mounted using prolonged gold antifade reagent (catalog no. p36930; Invitrogen) and visualized with confocal microscopy (Zeiss, Oberkochen,
Germany) using ZEN 2012 software. Images wereacquired using appropriate filter settings and were analyzed using ImageJ software for the quantification of
nuclear DAPI staining. The number of fluorescently

Habib et al. BMC Molecular and Cell Biology(2024) 25:5

stained nuclei was counted per field of view, and the average number of stained nuclei was calculated.
Propidium iodide (PI) staining and cell cycle analysis

After treatment, PC-3 cells were fixed for 30 min in 70%
ethanol at 4 °C. The cells were then washed twice in PBS,
and 100 µg/ml of RNase A (catalog no. 1,007,885; Qiagen) was added, followed by incubation for 20 min at
37 °C. The cells were then washed twice with PBS. Next,
the cells were incubated in 3 µM PI (catalog no. P4170;
Sigma) in staining buffer (100 mM Tris, pH 7.4, 150 mM
NaCl, 1 mM CaCl2, 0.5 mM MgCl2, 0.1% Nonidet P40)
for 15 min at room temperature. Cell cycle analysis was
carried out using a flow cytometer (Guava® easyCyte;
Millipore Sigma). Data acquisition and analysis were carried out using Guava cell cycle data acquisition and analysis software (Guava Technologies).
Annexin V and PI dual staining and flow cytometry analysis

PC-3 cells were harvested and washed twice with icecold phosphate-buffered saline (PBS). The cells werethen resuspended in annexin V binding buffer (10 mM
HEPES, pH 7.4, 140 mM NaCl, 2.5 mM CaCl2) at a concentration of 1 × 10^6 cells/ml. Annexin V-FITC conjugated (catalog no. A13199; Thermo Fisher) was added at adilution of 1:100, and the cells were incubated in the darkfor 15 min at room temperature.
Following Annexin V staining, the cells were washedtwice with the Annexin V binding buffer and resuspended in the same buffer. PI (final concentration of
3 µM) was added, and the cells were incubated in thedark for 15 min at room temperature. After PI staining,
the cells were washed twice with the annexin V binding buffer and fixed in 1% formaldehyde prepared in the
Annexin V binding buffer for 10 min on ice. The fixed
cells were then washed twice with PBS, and RNase A
was added at a final concentration of 100 µg/ml. The cells
were incubated for 20 min at 37 °C to digest RNA.
Prior to flow cytometry analysis, the cells were washedtwice with PBS and resuspended in 500 µL of PBS.
Annexin V/PI positivity and data analysis was conductedusing a flow cytometer (Guava® easyCyte; MilliporeSigma) and Guava data acquisition and analysis software(Guava Technologies).
Cell populations were classified into four categories
based on the staining pattern: viable cells (Annexin V-/PI-), early apoptotic cells (Annexin V+/PI-), late apoptotic or necrotic cells (Annexin V+/PI+), and necrotic
cells (Annexin V-/PI+). The percentage of cells in each
population was determined by gating on the appropriate
regions of the Annexin V/PI dot plot.

Page 4 of 14

Statistical analysis

Data were obtained from n independent biologicalexperiments and are presented as the mean of individual
values with standard deviation (SD) error bars or as boxand-whisker plots showing the median, the 25th and 75th
quartiles, as well as the minimum and maximum values.
To evaluate synergistic effects, the methodology established by Slinker et al. [24] was employed. Synergy wasdetermined based on the criterion that the combined
treatment effect (PTX + SFN) should surpass the cumulative effect of the individual drugs, as indicated by theequation:[Effect (PTX + SFN) > Effect (PTX) + Effect (SFN)]

Western blot densitometric analysis was carried out
using the ChemiDoc XRS system, Image Lab 6.0 (BioRad), and TotalLab TL120. GraphPad Prism 6 and Microsoft Excel software were used for statistical analyses andgraph generation.
Student’s t-test was used to compare two groups, andone-way analysis of variance (ANOVA) with Tukey’s correction was used for multiple comparisons to determine
the significant difference between PTX, SFN, PTX + SFN,
and the PC-3 non-stimulated cells (NS) that had notreceived any therapies.

Results
Cell viability, dose‑response analysis, and SFN effecton antiproliferation of PTX in PC‑3 cells

Different concentrations of PTX or SFN were prepared
as indicated in the Methods section, and the cells weretreated with the drugs.
MTT assay was conducted 24 h after treatment, andthe percentage of cell viability was determined for eachdrug concentration. Our data revealed that both PTX
and SFN significantly reduced PC-3 cell viability in adose-dependent manner (Fig. 1). ­TheEC50 value for PTX
was higher (1.2 mg/ml) than that for SFN-treated cells(18.7 µg/ml).
The SFN synergized PTX effect on reducing PC‑3 cell
viability

Our results showed that the combination treatment synergized the effects of both drugs on reducing cell viability. The ­EC50 value for the combination was 3.5 µg/ml,
which was 342-fold and 5.3-fold lower than the ­EC50 values for PTX and SFN individual treatments, respectively(Fig. 2A).
In a separate experiment, we compared the percentages of viable cells following treatment with PTX
or SFN individually and in combination. Our results
demonstrated that the PTX and SFN combination had

Habib et al. BMC Molecular and Cell Biology(2024) 25:5

Page 5 of 14

Fig. 1 shows the dose-response curves of PTX and SFN on PC-3 cell viability. The E­ C50 values for PTX and SFN were calculated, and the data
are presented as mean values with ± SD error bars. The MTT assay was used to measure cellular metabolic activity, and the data were obtained
from four independent experiments

Fig. 2 shows the synergistic effect of SFN and PTX on reducing cell viability in PC-3 cells. The E­ C50 value of the combined treatment
was determined by treating cells with equal concentrations of  PTX and SFN and conducting the MTT cell viability assay 24 h after treatment. The
data were obtained from five independent experiments and are presented as mean values with ± SD error bars in (A) or box-and-whisker plotsin (B). Student’s t-test was used to determine the significant difference between PTX and PTX + SFN or between SFN and PTX + SFN. The data showthat the combination treatment of SFN and PTX had a synergistic effect on reducing cell viability in PC-3 cells. (*p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001)

a significantly more potent effect on reducing PC-3
cell viability, even at a low concentration of 2 µg/ml.
At this concentration, the PTX and SFN combination
significantly reduced cell viability to 70.86% (p ≤ 0.013)
compared to the PC-3 non-stimulated cells, whichwere considered 100%. In contrast, at the same concentration of 2 µg/ml, neither PTX nor SFN individual treatments showed significant effects on reducing
the percentage of viable PC-3 cells (p ≥ 0.05) (Fig. 2B).
The effect of the combination treatment continued toHabib et al. BMC Molecular and Cell Biology(2024) 25:5

increase with increasing drug concentrations up to
8 µg/ml, and higher concentrations did not result infurther improvements in reducing cell viability.
A combination of PTX and SF Ninduces
Caspase‑3activation and nuclear morphology changes
characterizing apoptosis in PC‑3cells

Caspase 3 activation and changes in cell nuclei morphology, such as nuclear fragmentation and micronuclei appearance, are hallmarks of apoptotic cell death.
To evaluate the effect of PTX and/or SFN treatments on
inducing apoptosis in PC-3 cells, we treated the cells as
described in the Methods section. We utilized western
blot analysis and fluorescent microscopy to detect caspase 3 activation and nuclear morphological changes.
As expected, treatment with PTX or SFN resulted in
the cleavage of pro-caspase 3 protein into smaller active
caspase 3 subunits, which was detected by western blotat ~ 17 kDa. Densitometric analysis of caspase 3 protein
bands revealed that the intensity of cleaved caspase 3
bands in the protein lysate of PC-3 cells treated with thePage 6 of 14

PTX and SFN combination was significantly higher thanin the protein lysates of cells treated with PTX or SFN
individually (Fig. 3A).
In addition to caspase 3 activation, we observed significant nuclear morphology changes in PC-3 cells treatedwith the PTX and SFN combination. Specifically, weobserved nuclear fragmentation and micronuclei appearance, which are characteristic of apoptotic cell death.
These changes were detected using fluorescence microscopy and were not observed in cells treated with PTX or
SFN alone.
Microscopic visualization of DAPI-stained nuclei
showed morphological changes, including chromatincondensation, micronuclei, and nuclear fragmentation,
along with a noticeable reduction in the number of nuclei
in the visualized fields, likely due to cell detachment after
treatments. These changes were not observed in nonstimulated PC-3 cells, which retained a normal roundnuclei appearance (Fig. 3B). Furthermore, the apoptotic
cell death characteristics were more pronounced in cells
treated with the PTX + SFN combination compared

Fig. 3 shows that SFN additively enhances PTX-induced apoptosis in PC-3 cells. A Western blot and densitometric analysis of caspase-3
protein bands revealed significantly higher band intensities of cleaved caspase-3 in PTX + SFN-treated cells compared to PTX or SFN alone. This
was accompanied by a reduction in pro-caspase-3 bands. B Fluorescent micrographs of DAPI-stained nuclei showed nuclear fragmentationand micronuclei formation in cells treated with PTX, SFN, or PTX + SFN. The data were obtained from five independent experiments and arepresented as box-and-whisker plots for pro-caspase-3 and activecaspase-3 band intensities. Student’s t-test was used to determine the significant
difference between PTX or SFN individual treatments and PTX + SFN combined treatments. The data suggest that SFN enhances the apoptotic
effect of  PTX in PC-3 cells. (***p ≤ 0.001)

Habib et al. BMC Molecular and Cell Biology(2024) 25:5

to either agent alone. These findings suggest that SFN
enhances the apoptotic effect of PTX in PC-3 cells.
The combination effect of PTX and SFN on redistributing
the cell‑cycle growth phases in PC‑3

To investigate the effect of PTX and/or SFN on the cell
cycle growth phases in PC-3 cells, we stained the cellswith propidium iodide and analyzed them using flow
cytometry according to the methods section. Treatment with PTX or SFN increased the percentage of the
sub-G1 population by 9.23-fold (p ≤ 0.0002) or 9.10fold (p ≤ 0.0006), respectively, compared to non-stimulated cells. The effect of the combined treatment was
statistically more significant than PTX or SFN alone
and increased the sub-G1 population by 14.98-fold(p ≤ 0.0001) compared to non-stimulated cells. This
increase was 1.6-fold (p ≤ 0.003) and 1.7-fold (p ≤ 0.002)
higher than PTX or SFN alone, respectively (Fig. 4A). An
increasing sub-G1 population is indicative of apoptotic
cell death, and our data confirmed that this effect wasaugmented when cells received the PTX + SFN combined
treatment.
Furthermore, PTX or SFN induced an S-phase growtharrest by 6.38% (p ≤ 0.01) or 3.1% (p ≤ 0.05), respectively.
The combination treatment enhanced this effect to reach
9.93% (p ≤ 0.002).
To evaluate the necrotic effects of PTX and/or SFN,
we double-stained PC-3 cells with propidium iodide
and Annexin V and counted the necrotic cells using flow
cytometry. Our results showed that PTX or SFN treatments increased the number of necrotic cells by 5.23-fold(p ≤ 0.0002) or 5.74-fold (p ≤ 0.0003), respectively, compared to non-stimulated cells. Interestingly, there was no
significant difference among the fold-change values ofnecrotic cells in PTX, SFN, and the combination treatments (p ≥ 0.05). The combined treatment increased thenumber of necrotic cells to 5.71-fold (p ≤ 0.0002) compared to the number of necrotic cells in non-stimulated
PC-3 cells, which is similar to the fold-change values ofPC-3 cells treated individually with PTX or SFN (Fig. 4B).
The combination of PTX and SFN had a stronger effecton modulating Bax and ­Bcl2 protein expression

Protein lysates were prepared from the PC-3 cell line
and separated using SDS-PAGE. The protein bands
were then transblotted to nitrocellulose membranes andprobed with antibodies against Bax, Bcl2, and GAPDH.
The visualization and densitometric analysis of the bands
showed significant increases in Bax protein expression
by 185.08% (p ≤ 0.04) or 224.56% (p ≤ 0.01) following
PTX or SFN treatments, respectively. An additive effectwas observed on increasing Bax protein levels when cells

Page 7 of 14

were subjected to the combined treatment by 353.56%(p ≤ 0.0002). Bcl2 showed an opposite expression pattern
to Bax, where PTX or SFN reduced Bcl2 expression in
PC-3 cells. An additive effect was also observed in reducing Bcl2 levels after the combined treatments.
To further investigate, we calculated the ratio between
Bax and Bcl2 protein expression and found a significant increase in such ratios in PC-3 cells treated with
PTX or SFN compared to non-stimulated cells by 3.54fold (p ≤ 0.0007) or 3.4-fold (p ≤ 0.002), respectively. The
PTX + SFN combined treatment increased the Bax/Bcl2
ratio to 9.68-fold (p ≤ 0.0006) (Fig. 5A & B; Table S1).
To confirm our findings, we treated another prostatecancer cell line, LNCaP, with PTX, SFN, or the combination of PTX + SFN, as described in the methods section. The data collected from LNCaP cells confirmed that
PTX or SFN increased the protein expression of Bax andreduced Bcl2 levels, thus increasing the ratio of Bax/Bcl2.
These effects were augmented with the combined treatment of PTX + SFN (Fig. 5A & B; Table S2).

Discussion
Although boswellic acid, sulforaphane, and ginsenosidehave demonstrated remarkable anticancer activity andare being considered potential clinical candidates [25],it is worth noting that there are currently around 10,000
active clinical trials in the United States investigating
combination therapies for a range of conditions, including cancer, infectious diseases, metabolic disorders,cardiovascular diseases, autoimmune disorders, and neurological disorders.
Combination therapies are compared to single drugs
and are considered effective if they produce a better response when administered together than if they
are taken individually. However, because many drug
combinations could have additive or even synergistic effects, the degree of synergy becomes the mostimportant factor to consider. To answer this, the twodrug combination should be compared to not only single treatments but also the best of previously reported
drug combinations [26].
PTX has been used as an anticancer agent for treatinga variety of cancers since 1989; its therapeutic outcomes
did not meet the high expectations of physicians, rather
than patients; because many types of cancers, such as
prostate, breast, and ovarian cancers resist PTX treatment [27, 28]. In our endeavors to improve the anticancer potency of PTX and SFN, another promising andnaturally existing phytochemical found primarily ingreen cruciferous vegetables, may result in better treatment outcomes in prostate cancer cell models. Since
1987, when Levin and Hryniuk [29] introduced theconcept of drug dose intensity, the dose, and duration

Habib et al. BMC Molecular and Cell Biology(2024) 25:5

Page 8 of 14

Fig. 4 shows that SFN enhances PTX-induced cell-cycle arrest and apoptosis without affecting the number of necrotic PC-3 cells. The flow
cytometry analysis of cell-cycle growth phases demonstrated that PTX or SFN treatments increased the sub-G1 population, indicating
apoptotic cell death. The combination treatment of PTX and SFN showed a greater effect than either agent alone. The scatter plots of cells
double-stained with propidium iodide and Annexin V showed that both PTX and SFN treatments increased the number of necrotic cells compared
to non-stimulated cells. However, the combination treatment did not show a significant increase in the number of necrotic cells comparedto the individual treatments. These results suggest that SFN enhances the apoptotic effect of PTX in PC-3 cells without increasing the numberof necrotic cells. The data were obtained from five independent experiments and are presented as box-and-whisker plots for cell-cycle analysis
and scatter plots for apoptotic (A) and necrotic (B) effects. Significant differences were determined using the Student’s t-test for two-groupcomparisons. (***p ≤ 0.001)

of a given drug are precisely calculated to deliver the
maximum tolerated dose to overcome resistance tosuch chemotherapeutic agents. High drug doses aremost likely accompanied by severe side effects, which
is why researchers aspire to any therapeutic regimensthat would lead to lowering the dose and consequently
the side effects and maintaining the efficacy of the drug.
The data presented here showed that combining PTX
and SFN dramatically lowered the ­EC50 of both drugs
compared to the ­EC50 values of the same drugs whenHabib et al. BMC Molecular and Cell Biology(2024) 25:5

Page 9 of 14

Fig. 5 shows the effect of PTX, SFN, or PTX + SFN treatments on the expression of Bax and Bcl2 proteins in PC-3 and LNCaP cells. A The
Western blot and densitometric analysis histograms demonstrate significant differences between non-stimulated cells (NS) and PTX, SFN,
or PTX + SFN-treated cells. B The Bax:Bcl2 ratios in PC-3 and LNCaP cells are significantly increased with PTX, SFN, or PTX + SFN treatments. The
statistical analysis presented demonstrates the significance of the differences observed. The data provide further support for the additive effects
of PTX and SFN on inducing apoptosis in prostate cancer cells. (**p ≤ 0.01, ***p ≤ 0.001)

administrated individually. If applied to prostate cancer
patients, such findings may benefit them and help avoid
or attenuate the side effects of high doses.
Apoptosis is a natural process through which the bodyeliminates unwanted and damaged cells. A balanced
apoptosis/cell division rate protects from the occurrence of uncontrolled cell division and ultimately thedevelopment of cancers [30]. Unlike necrosis, apoptotic
cell death does not elicit an inflammatory response andallows the body to recycle the dead cell contents efficiently [31]. In cancer, chemotherapeutic drugs retrieve
the balance between apoptotic and survival signals by
specifically targeting cells with a high division rate. PTX
is known to halt mitotic division leading to cell cycle
arrest and apoptosis [32]; in our current study, we evaluated the effect of SFN on PTX-induced cell cycle arrest
and apoptosis in the PC-3 prostate cancer cell line.
Our data confirmed that SFN significantly improved

Habib et al. BMC Molecular and Cell Biology(2024) 25:5

PTX-induced activation of caspase-3 and increased
the number of accumulated cells in the S-growth phase(S-growth phase arrest). Such changes were accompaniedby the appearance of apoptotic bodies and micro-nuclei
in the treated cells. Interestingly, PTX + SFN combined
treatment did not increase the number of necrotic cells
comparable to PTX or SFN individual treatments. These
data indicate that SFN augmented the potency of PTX as
an anticancer agent and at the same time did not increase
necrosis, which is one of PTX’s side effects.
One of the strategies that a cancer cell adopts to evadeapoptosis is to upregulate the expression of survival signaling proteins, such as ­Bcl2, which showed higher levels
in prostate cancer [33, 34]. The elevated ­Bcl2 mRNA andprotein levels were specifically reported in the prostatecancer cell line (LNCaP) [34]. Higher levels of ­Bcl2 inhibit
caspase activities and result in resistance to apoptosis bypreventing the release of cytochrome c from the mitochondria [35]. Additionally, other researchers reported
that ­Bcl2 binds to the apoptosis-activating factor (APAF1) [36]; therefore, we proposed that lowering ­Bcl2 levels
may sensitize transformed cells to anticancer drugs, suchas PTX. The data presented here confirmed that PTX
in combination with SFN additively reduced the level of­Bcl2 protein compared with PTX or SFN alone in bothprostate cancer cell models (PC-3 and LNCaP). This
result is consistent with previously published reports,
where inhibiting Bcl2 using nonpeptide small molecule
inhibitors improved the therapeutic outcomes for targeting prostate cancer cells [37, 38]. Furthermore, the ratioof Bax (pro-apoptotic B­ cl2 family member): B­ cl2 protein
levels are crucial for cell survival [39], where increasing
Bax levels overcome the threshold that ­Bcl2 can neutralize, Bax, translocation to the mitochondria, leading to therelease of cytochrome c and trigger apoptosis [35]. So weinvestigated the levels of Bax protein following different
treatments and calculated the Bax:Bcl2 ratio. Our data
indicated that combination therapy of PTX and SFN significantly increased Bax protein expression and Bax:Bcl2
ratio compared to PTX or SFN individual treatments inboth PC-3 and LNCaP cell lines.
Limitations of the combination treatment of PTX and SFN
compared to individual treatments in both PC‑3 and LNCaP
cell lines

One limitation is the potential for increased toxicity
when two drugs are combined. While the combination
treatment of PTX and SFN was found to lower the E­ C50
of both drugs compared to individual treatments, thecombination treatment could lead to increased toxicity
in vivo. Therefore, careful dose optimization and toxicity
studies are needed to ensure that the combination treatment is safe and effective.

Page 10 of 14

Another limitation is the potential for drug resistance to develop with long-term use of the combination
treatment. While the combination treatment of PTX
and SFN has been shown to improve therapeutic outcomes in prostate cancer cell models, cancer cells could
develop resistance to the combination treatment overtime. Therefore, in clinical trials, it will be important tomonitor patients receiving this combination treatment
for signs of drug resistance and to develop strategies toovercome resistance if it develops.
Finally, it is important to note that the results of thisstudy were obtained in vitro, and the efficacy of the combination treatment of PTX and SFN in vivo may differ
from the results seen in cell culture. Therefore, further
preclinical studies and clinical trials will be needed todetermine the safety and efficacy of this combination
treatment in vivo.
The synergistic mechanisms through which SFN enhances
the efficiency of PTX

1. Highly effective anti-cancer therapy: Our findings
demonstrate that the combination of PTX and SFN
reduces resistance to PTX and allows for the use oflower doses of both PTX and SFN metabolites for
effective anti-cancer treatment as reported by Wang[40].
2. Enhanced Efficacy: Combining PTX with SFN has
demonstrated improved treatment outcomes and anincreased likelihood of tumor regression. The synergistic effect observed in the study resulted in a higher
level of apoptosis compared to individual treatments.
The combination therapy induced characteristic features of apoptotic cell death, such as nuclear fragmentation and the appearance of micronuclei. Thesemorphological changes indicate that the combination
treatment effectively promotes cancer cell death [41].
3. Caspase-3 Activation: Caspase-3 plays a crucial rolein the execution phase of apoptosis. Our results
indicated that the combination treatment of PTX
and SFN induces the activation of caspase-3. Caspase-3 activation leads to the cleavage of various target proteins, resulting in the dismantling of cellular
structures, causing microtubule degradation, andultimately leading to cell death, as evident in bothclinical and preclinical trials [3, 40, 41].
4. Cell Cycle Modulation: Treatment with PTX or SFN
alone increased the sub-G1 population, representing cells undergoing apoptotic cell death in human
bladder cancer T24 cells [42]. Our data demonstratethat the combination treatment further enhances thiseffect, indicating a higher rate of cancer cell death.
Moreover, both PTX and SFN individually induce

Habib et al. BMC Molecular and Cell Biology(2024) 25:5

S-phase growth arrest, and our current work confirms that the combination treatment amplifies thiseffect. S-phase arrest prevents cancer cells from replicating their DNA and progressing through the cell
cycle, ultimately leading to cell death [43].
5. Modulation of Bax and Bcl2 Protein Expression: Ourstudy reveals that PTX and SFN individually increase
the expression of Bax and decrease the expressionof Bcl2. This modulation of Bax and Bcl2 protein
expression promotes the pro-apoptotic signaling cascade, favoring apoptosis. An elevated Bax/Bcl2 ratio
signifies a shift towards pro-apoptotic signaling, leading to improved therapeutic outcomes [44, 45].
Because SFN-induced transcriptomic changes dependon the cell/tissue type, little is known about the context-dependent effects of SFN [46]. Thus, it is important to note that the precise mechanisms by which SFN
enhances PTX efficiency may vary depending on the specific cancer type and cellular context. Therefore, further
research is necessary to fully comprehend the mechanisms underlying the synergistic effects of SFN and PTX
in different types of cancer.
Are there any clinical trials investigating the efficacy of SFN
and PTX combination therapy?

There are currently no completed clinical trials investigating the efficacy of SFN and PTX combination therapy
in cancer patients. However, some ongoing clinical trials
are investigating the use of SFN and PTX in combination
with other treatments. Here are some examples:
1. A phase II clinical trial is currently investigating theefficacy of a combination of PTX, SFN, and cisplatinin patients with advanced non-small cell lung cancer(NSCLC) who have not received prior chemotherapy.
The study aims to evaluate the safety and efficacy ofthe combination treatment, and the primary endpoint is the overall response rate [47].
2. Another phase II clinical trial is investigating the efficacy of a combination of PTX, SFN, and carboplatinin patients with advanced ovarian cancer who havereceived prior platinum-based chemotherapy. The
study aims to evaluate the safety and efficacy of the
combination treatment, and the primary endpoint is
progression-free survival [48].
3. A phase I clinical trial is investigating the safety andtolerability of a combination of SFN and PTX in
patients with advanced solid tumors. The study aims
to determine the maximum tolerated dose of the
combination treatment, and the primary endpoint isdose-limiting toxicity [49].

Page 11 of 14

It should be noted that these clinical trials are currently
ongoing and their results are not yet available. Therefore,
it is unclear whether SFN and PTX combination therapy
will be effective in treating cancer patients in a clinicalsetting. However, these trials do suggest that SFN and
PTX combination therapy is a promising approach forcancer treatment, and further clinical studies are needed
to fully evaluate its safety and efficacy.
The potential side effects of SFN and PTX combination
therapy

They may include those associated with each drug individually, as well as potential synergistic effects.
1. Nausea and vomiting: Both PTX and SFN can cause
nausea and vomiting. PTX is known to be a highly
emetogenic drug, and SFN has been reported tocause nausea in some individuals [2, 50].
2. Neuropathy: PTX can cause peripheral neuropathy, which is characterized by numbness, tingling,
and pain in the hands and feet. SFN has been shownto have neuroprotective effects, but it is unclear
whether it can prevent or mitigate PTX-induced
neuropathy [51, 52].
3. Myelosuppression: PTX can cause myelosuppression,
which is a decrease in the number of blood cells produced by the bone marrow. SFN has been shown tohave hematopoietic effects, but it is unclear whether
it can prevent or mitigate PTX-induced myelosuppression [53, 54].
4. Liver toxicity: SFN has been shown to have hepatoprotective effects, but the combination treatment of
SFN and PTX could increase the risk of liver toxicity[55, 56].
5. Drug interactions: SFN has been shown to induce
phase II detoxification enzymes, which can increase
the metabolism of drugs metabolized by these
enzymes [57]. This could potentially decrease theefficacy of other drugs taken concurrently with PTX
and SFN, or increase the toxicity of these drugs.

Conclusion
The combination therapy of PTX and SFN holds great
promise for improving treatment outcomes in prostatecancer. This study demonstrated that the combination of
PTX and SFN resulted in a synergistic effect, significantly
lowering the effective concentrations of both drugs compared to individual treatments. This combination therapy
has the potential to benefit prostate cancer patients by
reducing side effects while maintaining drug efficacy.
The mechanism of action underlying the improved
therapeutic outcomes of the PTX and SFN combination therapy was investigated. The data showed that

Habib et al. BMC Molecular and Cell Biology(2024) 25:5

SFN improves the efficacy of PTX by enhancing apoptosis, activating caspase-3, inducing nuclear morphology changes, modulating the cell cycle, and altering theexpression of Bax and Bcl2 proteins. These findings provide mechanistic insights into the synergistic effects of
PTX and SFN, supporting the potential optimization of
combination therapy for prostate cancer treatment.
The preclinical evidence and mechanistic insights
gained from the PTX and SFN combination therapy
study provide a foundation for further research andclinical investigations. Future studies could explore the
combination therapy’s efficacy in different cancer types,
evaluate its potential in combination with other treatments, and investigate the underlying mechanisms invarious cellular contexts.
However, there are several limitations to consider.
Firstly, the potential for increased toxicity associated withthe combination treatment requires careful dose optimization and toxicity studies to ensure safety and effectiveness. Secondly, the development of drug resistance withlong-term use of combination therapy is a concern, highlighting the need for ongoing monitoring and strategies
to overcome resistance. Lastly, the results obtained invitro may not fully reflect the efficacy of the combination
treatment in vivo, necessitating further preclinical andclinical trials.

Page 12 of 14

Authors’ contributions
T.N.H, M.O.A, and M.S.S provided supervision, designed the study, analyzed thedata, and contributed to the discussion. M.A.H. contributed to the methodology and data collection. S.A.G provided writing review, editing, and data
analysis. All authors have reviewed and approved the final manuscript.
Funding
Open access funding provided by The Science, Technology & Innovation
Funding Authority (STDF) in cooperation with The Egyptian Knowledge
Bank (EKB). Open access funding is provided by a Transformative agreement
between Springer Nature and the Science, Technology & Innovation Funding
Authority (STDF) in cooperation with the Egyptian Knowledge Bank (EKB).
Availability of data and materials
The datasets used and/or analyzed during the current study are available fromthe corresponding author upon reasonable request.

Declarations
Ethics approval and consent to participate
Was granted by the Ethics Committee of Sohag University, College of Medicine, under code No. (90-06, Sohag, Egypt).
Consent for publication
Not applicable.
Competing interests
The authors declare no competing interests.
Author details
1
Molecular Genetics Lab, Department of Zoology, Faculty of Science, Sohag
University, Sohag, Egypt. 2 Snyder Institute for Chronic Diseases, Universityof Calgary, Calgary, AB T2N 4N1, Canada. 3 Department of Zoology, Facultyof Science, Ain Shams University, Cairo, Egypt.
Received: 2 August 2023 Accepted: 12 February 2024

Abbreviations
SFN	sulforaphane
PTX	Paclitaxel/ or Taxol
SFN	Sulforaphane
BPH	Benign prostatic hypertrophy
Nrf2	Nuclear factor-erythroid-2-related factor 2
PPAR-γ	Peroxisome proliferator-activatedγ
PSA	Prostate-specific antigen
HDAC3	Upregulated histone deacetylase protein
DMSO	Dimethyl sulfoxide
MTT	Thiazolyl blue tetrazolium bromide
RIPA	Radioimmunoprecipitation assay buffer
SDS-PAGE	Sodium dodecyl sulfate-Polyacrylamide gel electrophoresis
anti-GAPDH	anti-glyceraldehyde-3-phosphate dehydrogenase
DAPI	4’,6-diamidino-2-phenylindole
PBS	Phosphate-buffered salinePI	Propidium Iodide
APAF	Apoptosis-activating factor

Supplementary Information
The online version contains supplementary material available at https://​doi.​
org/​10.​1186/​s12860-​024-​00501-z.
Supplementary Material 1.
Acknowledgements
We gratefully acknowledge the Faculty of Science at Sohag University for providing the necessary chemicals and equipment for the experimental part ofthis study. We would like to extend our special thanks to Dr. Ali Fahmy, Professor of Molecular Toxicology at Vacsera Vaccination Centers, for providing thePC-3 cell lines, cell cycle analysis through the Flow Cytometry Laboratory, and
MTT assays. We sincerely appreciate his invaluable contributions to this study.

References
1. Zhang Y, Tang L. Discovery and development of sulforaphane as a cancer
chemopreventive phytochemical. Acta Pharmacol Sin. 2007;28(9):1343–
54. https://​doi.​org/​10.​1111/j.​1745-​7254.​2007.​00679.x.
2. Li Y, Zhang T, Korkaya H, Liu S, Lee HF, et al. Sulforaphane, a dietarycomponent of broccoli/broccoli sprouts, inhibits breast cancer stem cells.
Clin Cancer Res. 2010;16(9):2580–90. https://​doi.​org/​10.​1158/​1078-​0432.​
CCR-​09-​2937.
3. Singh AV, Xiao D, Lew KL, Dhir R, Singh SV. Sulforaphane induces
caspase-mediated apoptosis in cultured PC-3 human prostate cancer
cells and retards the growth of PC-3 xenografts in vivo. Carcinogenesis.
2004;25(1):83–90. https://​doi.​org/​10.​1093/​carcin/​bgg178.
4. El-Ashmawy NE, El-Zamarany EA, Khedr EG, et al. Combining paclitaxel
and sulforaphane for synergistic therapy against prostate cancer. Life Sci.
2022;289:120429.
5. Mattiuzzi C, Lippi G. Current Cancer epidemiology. J Epidemiol Glob
Health. 2019;9(4):217–22. https://​doi.​org/​10.​2991/​jegh.k.​191008.​001.
6. Singh O, Bolla SR. Anatomy, abdomen and pelvis, prostate. 2021. In:
StatPearls. Treasure Island: StatPearls Publishing; 2022. PMID: 31082031.
7. Kraemer PC, Borre M. Aflastning Af tumorbetinget obstruerede øvreurinveje ved prostate cancer [Relief of upper urinary tract obstruction inpatients with cancer of the prostate]. Ugeskr Laeger. 2009;171(11):873–6
Danish. PMID: 19278608.
8. Gann PH. Risk factors for prostate cancer. Rev Urol. 2002;4(Suppl 5):3-S10
PMID: 16986064; PMCID: PMC1476014.
9. Niclis C, Díaz Mdel P, Eynard AR, Román MD, La Vecchia C. Dietary habits
and prostate cancer prevention: a review of observational studies byfocusing on South America. Nutr Cancer. 2012;64(1):23–33. https://​doi.​
org/​10.​1080/​01635​581.​2012.​630163.

Habib et al. BMC Molecular and Cell Biology(2024) 25:5

10. Hu FB. Dietary pattern analysis: a new direction in nutritional epidemiology. Curr Opin Lipidol. 2002;13(1):3–9. https://​doi.​org/​10.​1097/​00041​433-​
20020​2000-​00002.
11. Niclis C, Román MD, Osella AR, Eynard AR, Díaz Mdel P. Traditional
Dietary Pattern increases risk of prostate Cancer in Argentina: results of aMultilevel modeling and Bias Analysis from a case-control study. J Cancer
Epidemiol. 2015;2015:179562. https://​doi.​org/​10.​1155/​2015/​179562.
12. Shinkai Y, Sumi D, Fukami I, Ishii T, Kumagai Y. Sulforaphane, an activatorof Nrf2, suppresses cellular accumulation of arsenic and its cytotoxicity inprimary mouse hepatocytes. FEBS Lett. 2006;580(7):1771–4. https://​doi.​
org/​10.​1016/j.​febsl​et.​2006.​02.​031.
13. Mahn A, Castillo A. Potential of sulforaphane as a natural Immune systemenhancer: a review. Molecules. 2021;26(3): 752. https://​doi.​org/​10.​3390/​
molec​ules2​60307​52.
14. Moi P, Chan K, Asunis I, Cao A, Kan YW. Isolation of NF-E2-related factor
2 (Nrf2), an NF-E2-like basic leucine zipper transcriptional activator thatbinds to the tandem NF-E2/AP1 repeat of the beta-globin locus control
region. Proc Natl Acad Sci USA. 1994;91(21):9926–30. https://​doi.​org/​10.​1073/​pnas.​91.​21.​9926.
15. Zhang Y, Talalay P, Cho CG, Posner GH. A major inducer of anticarcinogenic protective enzymes from broccoli: isolation and elucidation of
structure. Proc Natl Acad Sci USA. 1992;89(6):2399–403. https://​doi.​org/​
10.​1073/​pnas.​89.6.​2399.
16. Prochaska HJ, Santamaria AB, Talalay P. Rapid detection of inducers of
enzymes that protect against carcinogens. Proc Natl Acad Sci USA.
1992;89(6):2394–8. https://​doi.​org/​10.​1073/​pnas.​89.6.​2394.
17. Kwak MK, Wakabayashi N, Kensler TW. Chemoprevention through theKeap1-Nrf2 signaling pathway by phase 2 enzyme inducers. Mutat Res.
2004;555(1–2):133–48. https://​doi.​org/​10.​1016/j.​mrfmmm.
18. Brooks JD, Paton VG, Vidanes G. Potent induction of phase 2 enzymes
in human prostate cells by sulforaphane. Cancer Epidemiol. Biomarkers
Prev. 2001;10(9):949–54 PMID: 11535546.
19. Singh SV, Srivastava SK, Choi S, Lew KL, Antosiewicz J, Xiao D, Zeng Y,
Watkins SC, Johnson CS, Trump DL, Lee YJ, Xiao H, Herman-Antosiewicz
A. Sulforaphane-induced cell death in human prostate cancer cells is
initiated by reactive oxygen species. J Biol Chem. 2005;280(20):19911–24.
https://​doi.​org/​10.​1074/​jbc.​M4124​43200.
20. Cipolla BG, Mandron E, Lefort JM, Coadou Y, Della Negra E, Corbel L, Le
Scodan R, Azzouzi AR, Mottet N. Effect of sulforaphane in men with biochemical recurrence after Radical Prostatectomy. Cancer Prev Res (Phila).
2015;8(8):712–9. https://​doi.​org/​10.​1158/​1940-​6207.​CAPR-​14-​0459.
21. Beaver LM, Lӧhr CV, Clarke JD, Glasser ST, Watson GW, Wong CP, Zhang Z,
Williams DE, Dashwood RH, Shannon J, Thuillier P, Ho E. Broccoli sprouts
Delay prostate Cancer formation and decrease prostate Cancer severity
with a concurrent decrease in HDAC3 protein expression in transgenic
adenocarcinoma of the mouse prostate (TRAMP) mice. Curr Dev Nutr.
2017;2(3):nzy002. https://​doi.​org/​10.​1093/​cdn/​nzy002.
22. Bayat Mokhtari R, Homayouni TS, Baluch N, Morgatskaya E, Kumar S,
Das B, Yeger H. Combination therapy in combating cancer. Oncotarget.
2017;8(23):38022–43. https://​doi.​org/​10.​18632/​oncot​arget.​16723.
23. Weaver BA. How Taxol/paclitaxel kills cancer cells. Mol Biol Cell.
2014;25(18):2677–81. https://​doi.​org/​10.​1091/​mbc.​E14-​04-​0916.
24. Slinker BK. The statistics of synergism. J Mol Cell Cardiol. 1998;30(4):723–
31. https://​doi.​org/​10.​1006/​jmcc.​1998.​0655.
25. Tauro S, Dhokchawle B, Mohite P, Nahar D, Nadar S, Coutinho E. Natural
Anticancer agents: their therapeutic potential, challenges and Promisingoutcomes. Curr Med Chem. 2023. https://​doi.​org/​10.​2174/​09298​67330​
66623​05021​13150.
26. Rationalizing combination therapies. Nat Med. 2017;23(10):1113. https://​doi.​org/​10.​1038/​nm.​4426.
27. Rivera E, Gomez H. Chemotherapy resistance in metastatic breast cancer:
the evolving role of ixabepilone. Breast Cancer Res. 2010;12(2):2. https://​doi.​org/​10.​1186/​bcr25​73.
28. Gjyrezi A, Xie F, Voznesensky O, Khanna P, Calagua C, Bai Y, Kung J, Wu J,
Corey E, Montgomery B, Mace S, Gianolio DA, Bubley GJ, Balk SP, Giannakakou P, Bhatt RS. Taxane resistance in prostate cancer is mediated bydecreased drug-target engagement. J Clin Invest. 2020;130(6):3287–98.
https://​doi.​org/​10.​1172/​JCI13​2184.
29. Levin L, Hryniuk WM. Dose intensity analysis of chemotherapy regimensin ovarian carcinoma. J Clin Oncol. 1987;5(5):756–67. https://​doi.​org/​10.​
1200/​JCO.​1987.5.​5.​756.

Page 13 of 14

30. Elmore S. Apoptosis: a review of programmed cell death. Toxicol Pathol.
2007;35(4):495–516. https://​doi.​org/​10.​1080/​01926​23070​13203​37.
31. Nanji AA, Hiller-Sturmhöfel S. Apoptosis and necrosis: two types of cell
death in alcoholic liver disease. Alcohol Health Res World. 1997;21(4):325–
30 PMID: 15706744; PMCID: PMC6827678.
32. Karnak D, Xu L. Chemosensitization of prostate cancer by modulating
Bcl-2 family proteins. Curr Drug Targets. 2010;11(6):699–707. https://​doi.​
org/​10.​2174/​13894​50107​91170​888.
33. Chaudhary KS, Abel PD, Lalani EN. Role of the Bcl-2 gene family in prostate cancer progression and its implications for therapeutic intervention.
Environ Health Perspect. 1999;107(Suppl 1):49–57. https://​doi.​org/​10.​
1289/​ehp.​99107​s149.
34. Lin Y, Fukuchi J, Hiipakka RA, Kokontis JM, Xiang J. Up-regulation of
Bcl-2 is required for the progression of prostate cancer cells from anandrogen-dependent to an androgen-independent growth stage. Cell
Res. 2007;17(6):531–6. https://​doi.​org/​10.​1038/​cr.​2007.​12.
35. Yang J, Liu X, Bhalla K, Kim CN, Ibrado AM, Cai J, Peng TI, Jones DP, Wang
X. Prevention of apoptosis by Bcl-2: release of cytochrome c from mitochondria blocked. Science. 1997;275(5303):1129–32. https://​doi.​org/​10.​1126/​scien​ce.​275.​5303.​1129.
36. Haraguchi M, Torii S, Matsuzawa Si, Xie Z, Kitada S, Krajewski S, Yoshida H,
Mak TW, Reed JC. Apoptotic protease activating factor 1 (Apaf-1)-independent cell death suppression by Bcl-2. J Exp Med. 2000;191(10):1709–
20. https://​doi.​org/​10.​1084/​jem.​191.​10.​1709.
37. Wang S, Yang D, Lippman ME. Targeting Bcl-2 and Bcl-XL with nonpeptidic small-molecule antagonists. Semin Oncol. 2003;30(5 Suppl
16):133–42. https://​doi.​org/​10.​1053/j.​semin​oncol.​2003.​08.​015.
38. Korsmeyer SJ. Bcl-2: an antidote to programmed cell death. Cancer Surv.
1992;15:105–18 PMID: 1451107.
39. Edlich F, Banerjee S, Suzuki M, Cleland MM, Arnoult D, Wang C, Neutzner
A, Tjandra N, Youle RJ. Bcl-x(L) retro translocates Bax from the mitochondria into the cytosol. Cell. 2011;145(1):104–16. https://​doi.​org/​10.​1016/j.​cell.​2011.​02.​034.
40. Wang Y, Zhou Y, Zheng Z, Li J, Yan Y, Wu W. Sulforaphane metabolites
reduce resistance to paclitaxel via microtubule disruption. Cell Death Dis.
2018;9(11):1134. https://​doi.​org/​10.​1038/​s41419-​018-​1174-9.
41. Fimognari C, Berti F, Cantelli-Forti G, Hrelia P. Effect of sulforaphane on
micronucleus induction in cultured human lymphocytes by four different
mutagens. Environ Mol Mutagen. 2005;46(4):260–7. https://​doi.​org/​10.​
1002/​em.​20156.
42. Shan Y, Sun C, Zhao X, Wu K, Cassidy A, Bao Y. Effect of sulforaphane on
cell growth, G(0)/G(1) phase cell progression and apoptosis in human
bladder cancer T24 cells. Int J Oncol. 2006;29(4):883–8.
43. Rutz J, Thaler S, Maxeiner S, Chun FK, Blaheta RA. Sulforaphane reduces
prostate cancer cell growth and proliferation in vitro by modulating the
Cdk-Cyclin axis and expression of the CD44 variants 4, 5, and 7. Int J Mol
Sci. 2020;21(22):8724. https://​doi.​org/​10.​3390/​ijms2​12287​24.
44. Zhang L, Yu J, Park BH, Kinzler KW, Vogelstein B. Role of BAX in the apoptotic response to anticancer agents. Science. 2000;290(5493):989–92.
https://​doi.​org/​10.​1126/​scien​ce.​290.​5493.​989.
45. Tse C, Shoemaker AR, Adickes J, Anderson MG, Chen J, Jin S, Johnson EF,
Marsh KC, Mitten MJ, Nimmer P, Roberts L, Tahir SK, Xiao Y, Yang X, Zhang
H, Fesik S, Rosenberg SH, Elmore SW. ABT-263: a potent and orally bioavailable Bcl-2 family inhibitor. Cancer Res. 2008;68(9):3421–8. https://​doi.​
org/​10.​1158/​0008-​5472.​CAN-​07-​5836.
46. Etoh K, Nakao M. A web-based integrative transcriptome analysis, RNAseqChef, uncovers the cell/tissue type-dependent action of sulforaphane.
J Biol Chem. 2023;299(6): 104810. https://​doi.​org/​10.​1016/j.​jbc.​2023.​
104810.
47. ClinicalTrials.gov. A study of PTX, SFN, and cisplatin in patients withadvanced non-small cell lung cancer. 2023. https://​clini​caltr​ials.​gov/​ct2/​
show/​NCT03​705704.
48. ClinicalTrials.gov. A study of PTX, SFN, and carboplatin in patients withadvanced ovarian cancer. 2023. https://​clini​caltr​ials.​gov/​ct2/​show/​
NCT03​893514.
49. ClinicalTrials.gov. A study of sulforaphane and paclitaxel in advanced
solid tumors. 2023. https://​clini​caltr​ials.​gov/​ct2/​show/​NCT04​411058.
50. National Cancer Institute. Paclitaxel. 2023. https://​www.​cancer.​gov/​
about-​cancer/​treat​ment/​drugs/​pacli​taxel.

Habib et al. BMC Molecular and Cell Biology(2024) 25:5

51. National Cancer Institute. Peripheral Neuropathy. https://​www.​cancer.​
gov/​about-​cancer/​treat​ment/​side-​effec​ts/​neuro​pathy/​perip​heral-​neuro​
pathy-​fact-​sheet. Accessed 31 May 2023.
52. Kim HY, Kim SJ, Lee SM. Protective effect of sulforaphane against
paclitaxel-induced peripheral neuropathy in mice. Neurosci Lett.
2016;612:138–43.
53. National Cancer Institute. Myelosuppression. https://​www.​cancer.​gov/​
publi​catio​ns/​dicti​onari​es/​cancer-​terms/​def/​myelo​suppr​ession. Accessed
31 May 2023.
54. Hanlon N, Coldham N, Sauer MJ, et al. Induction of phase II detoxification
enzymes in rats by plant-derived isothiocyanates: comparison of allyl
isothiocyanate with sulforaphane and related compounds. J Agric Food
Chem. 2008;56(22):10551–9.
55. Kim YS, Ahn YH. New perspectives on the role of sulforaphane in Organic
Chemistry. Biomolecules. 2022;12(4):558.
56. Sung B, Chung HY, Kim ND. Role of apoptosis in Cancer Prevention andManagement. In: Bagchi D, Preuss HG, editors. Cancer Prevention andManagement through Exercise and Weight Control. Boca Raton (FL): CRC
Press/Taylor & Francis; 2005. Chapter 14.
57. Myzak MC, Tong P, Dashwood WM, Dashwood RH, Ho E. Sulforaphane
retards the growth of human PC-3 xenografts and inhibits HDAC activity
in human subjects. Exp Biol Med (Maywood). 2007;232(2):227–34 PMID:
17259330; PMCID: PMC2267876.

Publisher’s NoteSpringer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Page 14 of 14