Bisdemethoxycurcumin Augments Docetaxel Efficacy for Treatment of Prostate Cancer

Abstract

Bisdemethoxycurcumin (BDMC) is one of major forms of curcuminoids found in the rhizomes of turmeric. Docetaxel (DTX) is the standard of care for men diagnosed with androgen-independent prostate cancers. Here we report for the first time that BDMC could reinforce the effect of DTX against prostate cancer in vitro and in vivo. In vitro study, PC3 and LNCaP cells were cultured and treated with BDMC and DTX alone or in combination. The effects on cell viability were determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Apoptosis was assessed by annexin V/propidium iodide (PI) staining, while cell cycle was assessed by PI staining. Bax, Bcl-2, caspase, poly(ADP-ribose)polymerase (PARP), cyclin B1 and CDK1 expression were assayed by Western blot. We found that a combination treatment of BDMC (10 µM) with DTX (10 nM) was more effective in the inhibition of PC3 and LNCaP cell growth and induction of apoptosis as well as G2/M arrest, which is accompanied with the significant inhibition of Bcl-2, cyclin B1, CDK1 expression and significant increase of Bax, cleaved caspase-9, cleaved caspase-3 and cleaved PARP, than those by treatment of BDMC or DTX alone. Moreover, in vivo evaluation further demonstrated the superior anticancer efficacy of BDMC and DTX compared to DTX alone in a murine prostate cancer model. These results suggest that BDMC can be an attractive therapeutic candidate in enhancing the efficacy of DTX in prostate cancer treatment.

INTRODUCTION

Prostate cancer is a common urologic malignant tumor in men. It is the second most commonly diagnosed malignancy and the fifth leading cause of cancer-related mortality among men worldwide.¹⁾ Besides clinical and scientific efforts on prostate cancer, conventional therapies fail to eliminate advanced tumors. As prostate cancer cell growth is androgen dependent, its deprivation is an important therapeutic strategy. However, long-term androgen ablation results in androgen-independent cancer cell growth in metastatic patients, leading to castration-resistant prostate cancer (CRPC).²⁾ Currently, taxane-based chemotherapeutic drugs are the first line of treatment for CRPC.³⁾

Docetaxel (DTX), which has been used as a chemotherapeutic drug to combat recurrent prostate cancer, has demonstrated extraordinary anticancer effects in vitro and in vivo against a variety of tumors.^4–6) However, the use of high dose DTX always induced toxic reactions, and significant toxicity had precluded the use of DTX as a monotherapy for cancer.⁷⁾ In order to achieve higher antitumor efficacy and minimize the emergence of resistance, to search novel chemotherapy sensitizers become the focus in the field of cancer therapy. Recent studies are focused on combining nature-based agents with the conventional chemotherapies to augment the current cure rates in prostate cancer.^8,9)

Recent advances in the research of traditional Chinese medicine paved the way in the discovery of novel adjunct to chemotherapy.¹⁰⁾ Bisdemethoxycurcumin (BDMC) is a natural demethoxy derivative of curcumin which is also present in turmeric and there are accumulating evidences of the potent anti-cancer effects of BDMC.¹¹⁾ Compared with curcumin, which has the multifaceted chemopreventive potential,¹²⁾ only a few studies have been done to evaluate the beneficial effects of BDMC. Few studies have been carried out to investigate the underlying mechanisms regarding the beneficial effects of BDMC in cancer therapy. Moreover, BDMC was reported to have increased stability and improved nuclear cellular uptake compared to curcumin, strongly suggesting the potential of BDMC as an anti-cancer drug in clinical application.¹³⁾

The goal of our study is to elucidate whether BDMC could augment the efficacy of DTX in the treatment of prostate cancer. To assess the potential anticancer efficacy of BDMC and DTX, the human androgen-independent prostate cancer cells PC3 and androgen-dependent prostate cancer cells LNCaP was used to test in vitro cytotoxicity. Meanwhile, the in vivo antitumor efficiency of BDMC and DTX was evaluated in a murine prostate cancer model.

MATERIALS AND METHODS

Materials and Animals

Dulbecco’s modified Eagle’s medium (DMEM), fetal calf serum, trypsin, and antibiotics were purchased from Gibco (Grand Island, NY, U.S.A.). The RM-1 murine prostate cancer cell line, human nonmetastatic LNCaP and metastatic PC3 prostate cancer cells were obtained from ATCC (Manassas, VA, U.S.A.). In vitro tests, DTX was purchased from Sigma (St. Louis, MO, U.S.A.) and BDMC was obtained from Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan). Both DTX and BDMC were dissolved in dimethyl sulfoxide (DMSO) (Sigma) and diluted with media prior to the assays. All the primary antibodies were purchased from Santa cruze Technology (Danvers, MA, U.S.A.). In vivo tests, we have purchased DTX injection (Qilu Pharmaceutical Co., Ltd., Jinan, China) additionally for animal administration. All other chemicals were of analytical grade.

Animal experimental protocols (No. YTYW20230910-01) were approved by the Animal Use and Care Committee of Yantai Center for Food and Drug Control and were performed in agreement with National Institutes of Health Regulations on the scientific use of laboratory animals. C57BL/6 male mice (18–22 g, License No. SCXK 20220006) were purchased from Pengyue Animal Co., Ltd. (Jinan, China). Animals were given food and water available ad libitum, and were allowed to acclimatize for one week before the experiments.

Cell Culture

Prostate cancer cells (RM-1, PC3, and LNCaP) were grown in DMEM medium supplemented with 10% fetal bovine serum and 100 U/mL penicillin–streptomycin at 37 °C in a water-saturated atmosphere with 5% CO₂.

3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium Bromide (MTT) Assay

PC3 cells were plated at a density of 10⁴ cells/well in 96 well plates and allowed to adhere for 24 h prior to the assay.¹⁴⁾ Cells were exposed to a series of doses of BDMC and DTX at 37 °C. After 48 h of incubation, 50 µL of MTT indicator dye (5 mg/mL in phosphate-buffered saline, pH 7.4) was added to each well. Then the cells were incubated for 2 h at 37 °C in the dark. The formazan crystals were dissolved in 200 µL DMSO, and the optical density was measured at 570 nm using a microplate reader (SpectraMax M3, Molecular Devices, CA, U.S.A.). Growth inhibition was calculated as a percentage of the controls, which were not exposed to drugs. All experiments were repeated three times.

Apoptosis Assay

PC3 and LNCaP cells were seeded in 6-well plates at a density of 1 × 10⁵ per well. The cells were treated with vehicle, BDMC, DTX or combination of BDMC and DTX and after 48 h of treatment, the apoptosis was detected by annexin V apoptosis detection kit with propidium iodide.¹⁵⁾ Briefly, the cells were washed once with phosphate buffered saline (PBS), pellet was suspended in 100 µL of annexin V binding buffer followed by incubation with annexin V (conjugated with fluorescein isothiocyanate (FITC)) and propidium iodide (PI) in the dark for 15 min. The frequency of apoptotic cells was analyzed using flow cytometer (FACSAria, BD, NJ, U.S.A.).

Cell Cycle Assay

PC3 and LNCaP cells were plated at a density of 2 × 10⁵ per dish in 60-mm dishes. The cells were treated with vehicle, BDMC, DTX or combination of BDMC and DTX and after 48 h of treatment, cell cycle distribution was assessed by analyses of DNA content using a kit with adaptations.¹⁶⁾ Briefly, cells were collected and treated with 1 mL DNA labeling solution, containing detergent, PI and ribonuclease (RNase). Samples were then incubated in the dark for 10 min, at room temperature in a horizontal position and immediately analyzed by flow cytometry (FACSAria, BD).

Western Blot Analysis

After treatments, PC3 and LNCaP cells were placed in 10 cm dishes at a density of 1 × 10⁶ cells per dish. Cultured cells were washed with cold PBS and lysed in ice-cold lysis buffer (Beyotime, China) plus 1 : 100 volume of phenylmethylsulfonyl fluoride. Cell debris was removed after centrifugation (12000 × g, 4 °C, 10 min). After the protein concentration for each aliquot was determined by the Bradford method, suspensions were boiled in sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) loading buffer. Fifty microgram of the proteins obtained were separated using 12% SDS-PAGE gels. Blots were transferred to polyvinylidene fluoride membranes and incubated in blocking buffer (5% nonfat dried milk in Tris-buffered saline with Tween 20) at room temperature for 2 h, and then incubated with rabbit polyclonal antibody against relative protein (Bax, Bcl-2, Caspase-3, Cleaved caspase-3, Caspase-9, Cleaved caspase-9, poly(ADP-ribose)polymerase (PARP), cleaved PARP, CDK1 and cyclin B1) in diluent buffer overnight at 4 °C (1 : 1000 dilution) and anti-β-actin antibody, respectively. The membranes were washed with TBST and incubated with HRP-conjugated secondary antibody solution for 1 h at room temperature. The blots were washed thrice in TBST and detected by using enhanced chemiluminescence reagent and exposed to gel imaging (LAS4000, GE Healthcare, IL, U.S.A.). Images were collected and the bands corresponding to relative protein and β-actin protein were quantitated by densitometric analysis using the ImageQuant LAS4000 V1.22 software. Data of relative protein were normalized on the basis of β-actin levels.

In Vivo Studies

Murine prostate cancer model was prepared by subcutaneously injecting 2.0 × 10⁶ RM-1 cells, suspended in 0.1 mL of phosphate-buffered saline, into the armpit of the right anterior limb in C57BL/6 mice.¹⁷⁾ C57BL/6 mice were randomly divided into four matched groups: Control (Vehicle control; n = 7), BDMC 50 mg/kg (Vehicle + BDMC 50 mg/kg; n = 7), DTX 10 mg/kg (Vehicle + DTX 10 mg/kg; n = 7) and BDMC 50 mg/kg + DTX 10 mg/kg (Vehicle + BDMC 50 mg/kg + DTX 10 mg/kg; n = 7) groups. Except control group, the BDMC 50 mg/kg group received daily BDMC intragastrically at the dose of 50 mg/kg for 2 weeks. The DTX 10 mg/kg group received DTX injection intraperitoneally at the dose of 10 mg/kg for 2 weeks, twice a week. The combination group received BDMC and DTX injection simultaneously as mentioned above. Body weight was measured every 3 d. Mice were sacrificed 2 weeks after tumor inoculation and tumor tissues were isolated and captured, meanwhile the tumor weight was measured.

Statistical Analysis

To examine cell cycle experiments, we used Student’s t-test for paired data. Unless otherwise specified, the results of other experiments are presented as mean ± standard deviation (S.D.) of three independent experiments conducted in quadruplicate. The data were analyzed using one-way ANOVA followed by the Tukey’s multiple comparison test. The p < 0.05 was considered as significantly different.

RESULTS

BDMC Augments the Cytotoxic Effects of DTX in PC3 and LNCaP Cells

Both BDMC (2.5, 5, 10, 20, 40, and 80 µM) and DTX (1, 5, 10, 20, 50, 100, and 200 nM) used independently showed inhibitory effects on PC3 cell proliferation. BDMC (10 µM) and DTX (10 nM) were tested in combination, and found to be significant cytotoxicity for 48 h treated cells (Figs. 1A, B). The IC₅₀ values for DTX and BDMC were 34.1 and 33.6 µM for PC3 respectively at 48 h. A combined treatment of 10 µM BDMC and 10 nM DTX significantly decrease the proliferation compared to DTX and BDMC alone (Fig. 1C). Representative images of the PC3 cells are also shown respectively (Figs. 1D–G).

Fig. 1. BDMC Enhances the Anti-proliferative Effect of DTX on PC3 Cells

Cells were grown on 96 well plates and treated with sequential doses of either BDMC or DTX for 48 h. Thereafter, viability was measured by MTT assay (A, B) after 48 h. Cells were treated with either 10 nM DTX and 10 µM BDMC or combination of 10 nM DTX and 10 µM BDMC. Cell viability was measured by MTT assay after 48 h of treatment PC3 (C). Representative images of cells are shown above each treatment (D–G). Images were captured at 100× magnification. * p < 0.05 compared to BDMC or DTX group alone.

Both BDMC (2.5, 5, 10, 20, 40, and 80 µM) and DTX (1, 5, 10, 20, 50, 100, and 200 nM) used independently showed inhibitory effects on LNCaP cell proliferation. BDMC (10 µM) and DTX (10 nM) were tested in combination, and found to be significant cytotoxicity for 48h treated cells (Figs. 2A, B). The IC₅₀ values for DTX and BDMC were 35.5 and 34.3 µM for LNCaP respectively at 48 h. A combined treatment of 10 µM BDMC and 10 nM DTX significantly decrease the proliferation compared to DTX and BDMC alone (Fig. 2C). Representative images of the LNCaP cells are also shown respectively (Figs. 2D–G).

Fig. 2. BDMC Enhances the Anti-proliferative Effect of DTX on LNCaP Cells

Cells were grown on 96 well plates and treated with sequential doses of either BDMC or DTX for 48 h. Thereafter, viability was measured by MTT assay (A, B) after 48 h. Cells were treated with either 10 nM DTX and 10 µM BDMC or combination of 10 nM DTX and 10 µM BDMC. Cell viability was measured by MTT assay after 48 h of treatment LNCaP (C). Representative images of cells are shown above each treatment (D–G). Images were captured at 100× magnification. * p < 0.05 compared to BDMC or DTX group alone.

Therefore, we conducted experiments using 10 µM BDMC and 10 nM DTX.

BDMC in Combination with DTX Enhances Apoptosis in PC3 and LNCaP Cells

To confirm the effects of cotreatment with BDMC and DTX on PC3 and LNCaP cells, the cells were treated with the drugs for 48 h, cells were stained with annexin V-FITC and PI and analyzed by flow cytometry. On the flow cytometry charts, the Q1 represents necrotic cells, whereas, Q2, Q3, and Q4 refer to late apoptotic, early apoptotic respectively and viable cells. The BDMC (10 µM) and DTX (10 nM) apparently shifted the cells from Q4 to Q3 and Q2, which demonstrated the early and late apoptotic cells (Fig. 3). Furthermore, the combination treatment further increases the number of apoptotic cells. The early and late apoptotic cells increased up to 61.2 and 8.9% for PC3 treated with the combination, compared to 8.9 and 6.4% with BDMC, and 23.8 and 7.7% with DTX alone. The late apoptotic and necrotic cells increased up to 27.9 and 10.9% for LNCaP treated with the combination, compared to 9.3 and 3.1% with BDMC, and 4.2 and 0.4% with DTX alone.

Fig. 3. BDMC Enhances the DTX-Mediated Apoptosis in PC3 and LNCaP Cells

Cells were treated either with DTX 10 nM or BDMC 10 µM or a combination of DTX and BDMC for 48 h. Apoptosis was evaluated using Annexin V-FITC and PI staining followed by flow cytometry analysis. Percentage of early and late apoptotic cells and the necrotic cells are shown as numbers in bold in the flow cytometry chart. Q1 and Q2 represent necrotic and late apoptotic cells. Q3 and Q4 represent early apoptotic cells and viable cells respectively. The combination treatment significantly increased the proportion of PC3 (A) and LNCaP (B) cells in apoptotic and necrotic phase.

BDMC Augmented DTX-Induced G2/M Phase Cell Cycle Arrest

We determined the possible inhibitory effect of the combination of BDMC and DTX on PC3 and LNCaP cell cycle progression to investigate cell growth property of the combination. Compared with vehicle treatment, BDMC alone treatment for 48 h slightly increased the cell number arrested in S and G2/M. DTX alone treatment for 48 h moderately increased the cell number arrested in G2/M. Interestingly, the combination treatment resulted in a significant arrest in the G2/M phase in both cell lines (Fig. 4).

Fig. 4. Effect of the Combination Treatment of BDMC and DTX on the Cell Cycle Progression of PC3 and LNCaP Cells

Cells were treated either with DTX 10 nM or BDMC 10 µM or a combination of DTX and BDMC for 48 h, and then the proportion of cells at different cell cycle phases was analyzed by flow cytometry. The combination treatment significantly increased the proportion of PC3 (A) and LNCaP (B) cells in G2/M phase. The typical histograms were also shown and the data were expressed as the mean ± S.D. of three independent experiments. * p < 0.05 compared to BDMC or DTX group alone.

BDMC Augmented DTX-Induced the Expression of Apoptotic Regulatory Proteins

To identify the mechanism of enhanced PC3 and LNCaP cells growth inhibition and apoptotic response by BDMC and DTX combination treatment, we next assessed the expression of apoptotic regulatory proteins by Western blot. The combination treatment substantially inhibited the expression of all marker proteins tested that favored cell survival and anti-apoptotic protein, but enhanced the expression of pro-apoptotic proteins. As shown in Fig. 5 (PC3 cells) and Fig. 6 (LNCaP cells), expression of Bax, cleaved caspase-3 (17 and 19 kDa) and cleaved caspase-9 as well as cleaved PARP were much greatly increased by the treatment of the combination of BDMC and DTX compared to that by single treatment. Our data also showed that the combination treatment significantly inhibited expression of Bcl-2 expression compared to that by single treatment. Additionaly, the effect of the combination treatment on cell cycle regulatory molecules operative in G2/M phase of the cell cycle was determined. The expression of G2/M regulators cyclin B1 and CDK1 were analyzed. The treatment with the combination for 48 h resulted in a significant reduction in cyclin B1 and CDK1 expression.

Fig. 5. Effect of the Combination Treatment of BDMC and DTX on Expression of Apoptosis as Well as Cell Cycle Regulatory Proteins in PC3 Cells

(A) PC3 cells were co-treated with 10 µM BDMC and 10 nM DTX for 48 h. Expression of Bax, Bcl-2, Caspase9, Cleaved caspase9, Caspase3, Cleaved caspase3, PARP and cleaved PARP were detected by Western blot using specific antibodies. (B) Effect of BDMC or DTX or the combination on the expression of cell cycle related proteins (cyclin B1 and CDK1) was determined using Western blot. The cells were treated with or without BDMC or DTX or the combination for 48 h. β-Actin was used as an internal control. The data were expressed as the mean ± S.D. of three independent experiments. * p < 0.05, ** p < 0.01 compared to BDMC or DTX group alone.

Fig. 6. Effect of the Combination Treatment of BDMC and DTX on Expression of Apoptosis as Well as Cell Cycle Regulatory Proteins in LNCaP Cells

(A) LNCaP cells were co-treated with 10 µM BDMC and 10 nM DTX for 48 h. Expression of Bax, Bcl-2, Caspase9, Cleaved caspase9, Caspase3, Cleaved caspase3, PARP and cleaved PARP were detected by Western blot using specific antibodies. (B) Effect of BDMC or DTX or the combination on the expression of cell cycle related proteins (cyclin B1 and CDK1) was determined using Western blot. The cells were treated with or without BDMC or DTX or the combination for 48 h. β-Actin was used as an internal control. The data were expressed as the mean ± S.D. of three independent experiments. * p < 0.05, ** p < 0.01 compared to BDMC or DTX group alone.

BDMC Augmented DTX-Induced Antitumor Efficacy in Vivo Studies

Antitumor efficacy of BDMC and DTX, when singly or in combination, was investigated in a murine prostate cancer model for 2 weeks. The study was to evaluate whether the combination treatment of BDMC and DTX could generate superior antitumor activity compared with single administration of either drug. The combination of BDMC and DTX inhibited the growth of tumor more efficiently than single delivery of BDMC or DTX (Fig. 7A). The growth of tumors among the different treatment groups were shown, which could be observed clearly that the tumors from the mice treated with DTX and BDMC were obviously smaller than those of other groups (Fig. 7B). An analysis of body weight variation generally defined the adverse effects of the different therapy. No significance was observed between the combination group and DTX group alone (Fig. 7C).

Fig. 7. Variation of Body Weight, Tumor Weight and Tumor Images during Therapy in Vivo Studies

(A) Body weight change of C57BL/6 mice with different treatments during therapy. (B) Tumor weight change of C57BL/6 mice with different treatments at the end of the experiment. (C) The images of excised tumors at the time of sacrifice from the C57BL/6 mice after 2 weeks of therapy. The data were expressed as the mean ± S.D. * p < 0.05 compared to BDMC or DTX group alone.

DISCUSSION

Prostate cancer has become an increasingly prevalent malignancy in males worldwide.¹⁸⁾ Recent studies have shown that curcumin has potent antitumor effects on prostate cancer.^19,20) Compared with curcumin, which can be easily degraded both in vitro and vivo, the structure of BDMC lacks two methoxy group directly linking to the benzenering, is more stable. Moreover, the combination therapy with natural compounds is being popular because of the minimum adverse effects on healthy cells.²¹⁾ In agreement, the present study indicates that BDMC strongly augments the inhibitory effect of DTX on prostate cancer. Our study suggests that BDMC considerably enhances the efficacy of DTX treatment in PC3 and LNCaP cells by inhibiting proliferation and inducing apoptosis through modulation of related proteins compared to DTX or BDMC alone.

The current study clearly demonstrated that the activation of mitochondria-mediated apoptotic pathway was involved in the combination effect of BDMC and DTX treatment in PC3 and LNCaP cells. We analyzed the expression of Bcl-2 and Bax, playing an antiapoptotic and proapoptotic role, respectively. Bax, normally residing in the cytosol, translocates to mitochondria to promote apoptosis, but Bax activity is counteracted by antiapoptotic proteins, such as Bcl-2.^22,23) Caspase activation serves an indispensable function in the execution of mitochondria-mediated apoptosis.^24,25) Bax targets the mitochondria to antagonize Bcl-2 effect and promotes the release of cytochrome C.²⁶⁾ Release of cytochrome C in the cytoplasm activates caspase-9, activated caspase-9 cleaves and activates caspase-3 in a cascade that subsequently cleaves DNA repair protein PARP, leading to the nuclear cleavage.^27–29) Our data showed that consistent with the increase of apoptosis, the expression of apoptotic proteins active caspase-3 and Bax was much more increased by the combination of BDMC and DTX treatment compared to that by BDMC or DTX alone treatment. However, the expression of Bcl-2 by the combination was much lower than that by BDMC and DTX alone treatment. Thus, the alteration of the anti- and pro-apoptotic protein levels is likely to influence the combination effect of the BDMC and DTX on the inhibition of PC3 and LNCaP cell growth.

Molecular analyses of human cancers have revealed that cell cycle regulators are frequently involved in most of the common malignancies.^30,31) The eukaryotic cell cycle is regulated by cyclins and cyclin-dependent kinases.³²⁾ In eukaryotes, the decreased CDK1/Cyclin B1 complex formation correlates with G2/M phase accumulation.^33,34) Our cell cycle analysis revealed that consistent with the greater effect on the induction of apoptosis, much more cells were arrested in the G2/M phase by the combination treatment of BDMC and DTX. Each drug contributes to cell cycle arrest in agreement with the observed anti proliferative effect. However, DTX induces G2/M arrest, while BDMC induces G2/M and S arrest. In addition, remarkably greater changes of the expression of cell cycle related proteins, cyclin B1 and CDK1 were observed in the cells treated with the combination. In this study, the combination of BDMC and DTX resulted in G2/M arrest, decreased CDK1 and Cyclin B1 protein expressions, leading to the blockade of cell cycle progression in PC3 and LNCaP cells.

The tumor inhibition efficacy toward the animal model is an essential method for evaluating a controlled drug delivery system.³⁵⁾ Since RM-1 cells are a murine prostate cancer cell line, the RM-1 model possesses some distinct characteristics, such as rapid tumor growth and high aggressiveness, which is especially suitable for anti-cancer studies.³⁶⁾ In vivo, the inhibition effect on growth of murine prostate tumor was much greater by the combination of BDMC (50 mg/kg) and DTX (10 mg/kg) treatment compared to that by BDMC or DTX alone treatment. Moreover, simultaneous administration of BDMC and DTX shows little side-effect through the variation of body weight. Our data demonstrated that no significance was observed between the combination group and DTX group alone in the variation of body weight.

In conclusion, results from this work offer an effective way to improve the anticancer efficiency of DTX. BDMC could be useful as an anticancer adjuvant agent in the combination chemotherapy. These findings suggest that BDMC and DTX combination may be more effective on prostate cancer treatment than DTX alone. However, the development of BDMC as drug sensitizers needs more intensive research in order to evaluate the feasibility and advantages of clinical applications.

Conflict of Interest

The authors declare no conflict of interest.

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