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 Table of Contents  
Year : 2018  |  Volume : 4  |  Issue : 5  |  Page : 117-122

Enhanced anticancer effect by combination of proteoglucan and Vitamin K3 on bladder cancer cells

Department of Urology, New York Medical College, Valhalla, NY, USA

Date of Web Publication30-Oct-2018

Correspondence Address:
Dr. Sensuke Konno
Department of Urology, New York Medical College, BSB Room A03, Valhalla, NY 10595
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/ctm.ctm_25_18

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Aim: Mushroom extract, PDF, is a bioactive proteoglucan with anticancer/antitumor activity, and Vitamin K3 (VK3) is a synthetic VK derivative with antitumor activity as well. An unconventional approach using these two agents was tested to see their anticancer effects on bladder cancer cells in vitro.
Methods: Human bladder cancer T24 cells were treated with PDF, VK3, or their combination, and cell viability was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide assay. To explore the anticancer mechanism, cell cycle and epigenetic alterations were specifically studied.
Results: PDF ≥ 500 μg/mL led to a ~ 35% reduction in cell viability while VK3 had little effects. However, when PDF (300 μg/mL) was combined with VK3 (5 μM), a ~ 75% cell viability reduction was attained. This specific combination induced a G1 cell cycle arrest with the downregulation of G1-specific regulators. In addition, histone deacetylase was inactivated while histones 3 and 4 were highly acetylated. Two apoptotic regulators were significantly activated with PDF/VK3 combination as well.
Conclusion: The specific combination of PDF and VK3 appears to potentiate anticancer effect on T24 cells. This is primarily attributed to a G1 cell cycle arrest with chromatin modifications, ultimately leading to apoptosis. Thus, the PDF/VK3 combination may offer a potential therapeutic option for bladder cancer.

Keywords: Anticancer, apoptosis, bladder cancer, proteoglucan, Vitamin K3

How to cite this article:
Zhang M, Zheng K, Choudhury M, Phillips J, Konno S. Enhanced anticancer effect by combination of proteoglucan and Vitamin K3 on bladder cancer cells. Cancer Transl Med 2018;4:117-22

How to cite this URL:
Zhang M, Zheng K, Choudhury M, Phillips J, Konno S. Enhanced anticancer effect by combination of proteoglucan and Vitamin K3 on bladder cancer cells. Cancer Transl Med [serial online] 2018 [cited 2018 Nov 21];4:117-22. Available from: http://www.cancertm.com/text.asp?2018/4/5/117/244518

  Introduction Top

Bladder cancer is the second common genitourinary malignancy next to prostate cancer in the United States.[1] Nearly 82,000 new cases would be diagnosed and ~ 17,000 people may die this year (2018).[1] Approximately 75% of all newly diagnosed cases will present nonmuscle invasive bladder cancer (NMIBC) including stage Ta, T1 and carcinoma in situ,[2],[3] while other 25% are muscle invasive bladder cancers (MIBCs) or metastatic cancers.[3] The primary treatment for NMIBC is transurethral resection of bladder tumor (TURBT);[4] however, NMIBC following TURBT has a high recurrence rate of up to 70%, of which ~ 20% may progress to advanced MIBC.[5] The reason(s) for this high recurrence is/are not fully understood, but a multifocal origin of disease, as well as intrinsic acquired resistance to drugs, appears to be a major cause of such a high incidence of disease relapse.[6] Hence, the primary therapeutic aim is to prevent multiple recurrences and progression to a more advanced, MIBC.

To reduce or minimize the risk of tumor recurrence, intravesical immunotherapy with bacillus Calmette–Guerin (BCG), an attenuated strain of Mycobacterium bovis, is often recommended following TURBT.[5],[7] This intravesical BCG administration is indeed the most effective therapy among currently available therapeutic options – it has been shown to alter disease progression, reduce recurrence, and increase survival.[7],[8] However, up to 80% of patients will have recurrence and up to 45% of them may progress to MIBC within 5 years.[9] In addition, its benefits are also outweighed by its severe side effects including cystitis, fever, sepsis, and allergic reactions[10] so that BCG treatment is often withdrawn from the recommended 1–3 year treatment protocol. These concerns thus limit its use in clinical practice and demonstrate the need for a nontoxic, safe, effective treatment modality with few side effects.

Vitamins have been long known as the beneficial nutritional supplements, and their role in cancer prevention and treatment has also been extensively studied.[11],[12],[13] Among them, we were particularly interested in Vitamin K3 (menadione; VK3), a synthetic derivative of naturally occurring fat-soluble VK. It has been initially shown to be involved in blood coagulation[12] and also inhibit the growth of several tumor cells.[13] Such antitumor potency of VK3 was found to be far greater than VK1 and VK2, which were other derivatives of VK.[12] Although VK3 by itself may have antitumor activity, it yet requires a high dose to be effective.[12],[13] To compensate this drawback, combinations of VK3 and other vitamins, drugs, or agents have been attempted to improve its efficacy on cancer cells at its relatively lower doses.

Meanwhile, we have been exploring a more effective therapeutic modality for bladder cancer using natural agents/substances. These include herbs, mushrooms, flowers, fruits, plant seeds, sea weeds, algae, tea, bark, shark cartilage, etc. Although most of them lack scientific demonstrations, maitake (Grifola frondosa), one of medicinal mushrooms, has been extensively studied.[14] Its antitumor activity has been shown in tumor-bearing mice,[15] through activation of various immune effectors, such as macrophages, cytotoxic T-lymphocytes, and natural killer cells.[16] Moreover, the bioactive extract of this mushroom known as proteoglucan (PDF) has been commercially available for scientific studies or personal consumption.

Accordingly, as an unconventional approach, we investigated if VK3 or PDF by itself might have anticancer effect or their combination would further enhance it on bladder cancer cells in vitro. To understand how they work, the anticancer mechanism was also explored, focusing specifically on the cell cycle regulation and epigenetic modification involving histone deacetylase (HDAC) and histones (H3 and H4).[17],[18],[19] More details and the interesting findings are described herein.

  Methods Top

Cell culture

The human bladder cancer T24 cells were obtained from the American type culture collection (ATCC; Manassas, VA, USA). Cells were cultured in RPMI 1640 medium containing 10% fetal bovine serum, penicillin (100 U/mL), and streptomycin (100 μg/mL). For experiments, cells were seeded in six-well plates or T-75 flasks at the initial cell density of 2 × 105 cells/mL and were cultured with standardized proteoglucan (PDF; Mushroom Wisdom, Inc., East Rutherford, NJ, USA), VK3 (Sigma-Aldrich, St. Louis, MO, USA), or their combinations. Cell viability was then assessed using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) assay below.

3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide assay (cell viability test)

Cell viability was assessed by the MTT assay following the vendor's protocol (Sigma-Aldrich, St. Louis, MO, USA). Briefly, at the harvest time, 1 mL of MTT reagent (1 mg/mL) was added to each well in the 6-well plate, followed by 3-h incubation at 37°C. After removing MTT reagent, dimethyl sulfoxide was added to each well and absorbance of formazan solution (purple) was read on a microplate reader. Cell viability was then determined and expressed by the percentage (%) of viable cells relative to the control reading (100%). All experiments were repeated three times to perform statistical analysis, which will determine the mean value and standard deviation for assessing the significance in the differences between groups tested.

Cell cycle analysis

A FACScan flow cytometer (Becton Dickinson, San Jose, CA, USA), equipped with a double discrimination module, was employed for cell cycle analysis. Control or agents-treated cells (~ 1 × 106 cells per condition) were first resuspended in 500 μL of propidium iodide solution and incubated for 1 h at room temperature in the dark. Following incubation, ~ 10,000 nuclei from each sample were analyzed on a flow cytometer, and CellFit software (Becton Dickinson, San Jose, CA, USA) was used to quantify cell cycle compartments to estimate the percentage of cells distributed in the different cell cycle phases. All experiments were repeated three times separately to perform statistical analysis.

Western blot analysis

An equal amount of proteins (10 μg) from control and agent-treated cell lysates was resolved by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to a nitrocellulose membrane (blot). The blot was first incubated for 90 min with the primary antibodies against CDK2, cyclin E, p21Waf1, HDAC1, or acetylated H3/H4 (AcH3/H4) (Santa Cruz Biotechnology, Santa Cruz, CA, USA), followed by incubation with the secondary antibody conjugate for 30 min. The immunoreactive protein bands were detected by chemiluminescence following the manufacturer's protocol (Kirkegaard and Perry Laboratories, Gaithersburg, MD, USA) and quantified using a scan densitometer (Silk Scientific, Orem, UT, USA). This analysis was repeated at least three times to confirm reproducibility.

Enzymatic assays for caspase-3 and caspase-9

Enzymatic activities of caspase-3 (Csp-3) and caspase-9 (Csp-9)[20],[21] were determined following the colorimetric method described in the vendor's protocol (BioVision, Milpitas, CA, USA). An equal amount (100 μg) of cell lysates prepared from control or treated cells was incubated with the reaction mixture containing the substrates, DEVD-pNA (for Csp-3) or LEHD-pNA (for Csp-9), at 37°C for 2 h. Absorbance of the reaction mixture was read spectrophotometrically using a microplate reader. Fold-increases in Csp-3/9 activities were then calculated by comparing the readings of treated cells with the controls (an arbitrary value of 1). This enzymatic assay was repeated three times and subjected to statistical analysis.

Statistical analysis

All data are presented as the mean ± standard deviation, and statistical differences between groups are assessed with either the unpaired Student's t-test or one-way ANOVA analysis. P < 0.05 is considered to indicate statistical significance.

  Results Top

Possible anticancer effects of PDF or Vitamin K3 on T24 cells

As anticancer effect can be assessed by cell viability, i.e., the percentage of viable cells, T24 cells treated separately with PDF (0–700 μg/mL) or VK3 (0–8 μM) for 72 h were subjected to MTT assay. The results showed that PDF would induce a ~ 20% and ~ 35% reduction in cell viability at 500 and 700 μg/mL, respectively [Figure 1]a. However, no significant effects of VK3 at any given concentrations were seen on cell viability [Figure 1]b. These results thus show that generally PDF and VK3 alone at the relatively low concentrations have little anticancer effects.
Figure 1: Effects of PDF or Vitamin K3on T24 cell viability. Cells were cultured with the varying concentrations of either PDF (0–700 μg/mL) or Vitamin K3 (0–8 μM), and cell growth with PDF (a) or Vitamin K3 (b) at 72 h was assessed by the percentage of viable cell numbers relative to that in control (100%). All data represent mean ± standard deviation from three independent experiments (*P < 0.05 compared with control)

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Enhanced anticancer effects of combinations of PDF and Vitamin K3

Although individual PDF and VK3 appear to have little effects, it was tempting to examine whether combinations of PDF and VK3 might exhibit anticancer effect. Cells were treated with ineffective PDF (300 μg/mL) [Figure 1]a and ineffective VK3 (1, 3, or 5 μM) [Figure 1]b together and cell viability was determined in 72 h. The results showed that combinations of PDF (300 μg/mL) and VK3 at 3 and 5 μM led to a ~ 40% and ~ 75% cell viability reduction, respectively [Figure 2]. Thus, this study demonstrates that the PDF/VK3 combinations can exhibit the enhanced anticancer effect, resulting in the significant cell viability reduction.
Figure 2: Effects of combination of PDF and Vitamin K3on cell viability. Cells were treated with combinations of PDF (300 μg/mL) and 1, 3, or 5 μM of Vitamin K3for 72 h, and cell viability was assessed by the percentage of viable cell numbers relative to controls (100%). The data are mean ± standard deviation from three separate experiments (*P < 0.05 compared with control)

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Effects of PDF and Vitamin K3 on cell cycle

To explore the anticancer mechanism of the PDF/VK3 combination, cell cycle analysis was performed on T24 cells treated with the most effective combination of PDF (300 μg/mL) and VK3 (5 μM). After 72 h, compared to controls, the PDF/VK3 combination induced a 54% increase and 52% decrease in G1 and S phase cell numbers, respectively [Figure 3], although PDF or VK3 alone had little effects. This cell accumulation in the G1 phase is known as a G1 cell cycle arrest.,[22] i.e., a halt of cell cycle transition at the G1 phase. Thus, the PDF/VK3 combination may interfere with cell cycle, eventually leading to the growth cessation and even cell death.
Figure 3: Cell cycle analysis. Cells were treated with PDF (300 μg/mL), Vitamin K3 (5 μM), or their combination for 72 h, followed by cell cycle analysis to evaluate the effects of these agents on distribution of cell populations in specific cell phases. All data are mean ± standard deviation from three separate experiments but only those mean values are shown here for a better comprehension (*P < 0.05 compared to control)

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Effects of PDF/Vitamin K3 combination on G1 cell cycle regulators

To confirm a PDF/VK3-induced G1 cell cycle arrest, the status of the G1-specific cell cycle regulators was also examined.[22] Cells treated with or without the combination of PDF (300 μg/mL) and VK3 (5 μM) for 72 h were analyzed for CDK2, cyclin E, and p21Waf1 on Western blots. Such analysis revealed that compared to controls, the expressions of CDK2 and cyclin E were significantly reduced or downregulated with the PDF/VK3 treatment [Figure 4]a. In contrast, p21Waf1 known to block the G1-S phase transition[23] was enhanced or upregulated in PDF/VK3-treated cells [Figure 4]a. The intensity/density of those protein bands was also quantified for a better understanding [Figure 4]b. Thus, these results would provide the further evidence that the PDF/VK3 combination indeed leads to a G1 cell cycle arrest.
Figure 4: Effects of PDF/Vitamin K3combination on specific cell cycle regulators. Cells treated with or without the PDF (300 μg/mL)/Vitamin K3 (5 μM) combination for 72 h were analyzed for the expressions of G1-specific cell cycle regulators by Western blots. (a) Autoradiographs of CDK2, cyclin E, and p21Waf1 in control and PDF/Vitamin K3-treated cells are shown for comparison. (b) The density of the protein bands are also quantified by scan densitometry and plotted in the graph (*P < 0.05 compared with respective control)

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Effects of PDF/VK3-induced chromatin modifications

As activation of p21Waf1 is mediated through hyperacetylation of histones in the chromatin,[17],[18] we examined if the PDF/VK3 combination might have any effects on three epigenetic parameters, HDAC1, AcH3, and AcH4, using Western blots. After 72-h treatment with or without the PDF/VK3 combination, the expression of HDAC1 was significantly reduced or inactivated while those of AcH3 and AcH4 were elevated or hyperacetylated with PDF/VK3 treatment [Figure 5]a. Those protein bands were also quantified for a clear comparison [Figure 5]b. These results suggest that inactivation of HDAC1 induces hyperacetylation of H3/H4,[19] which then activates p21Waf1 (leading to a G1 cell cycle arrest).[23] This is consistent with the established relationship between HDAC1, H3/H4, and p21Waf1.[17],[18] It should be yet noted that PDF or VK3 alone had no effects on all these parameters. Thus, only the PDF/VK3 combination may induce chromatin modifications, subsequently leading to a G1 cell cycle arrest.
Figure 5: Chromatin modifications. Cells treated with or without the PDF (300 μg/mL)/Vitamin K3 (5 μM) combination for 72 h were analyzed for three epigenetic parameters using Western blots. (a) Autoradiographs of histone deacetylase 1, acetylated H3, and acetylated H4 in control and PDF/Vitamin K3-treated cells are shown for comparison. (b) The density of the protein bands quantified by scan densitometry are plotted in the graph (*P < 0.03 compared with respective control)

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Induction of apoptosis

As a G1 cell cycle arrest could often cause cell death, it was important to address if such cell death might be linked to apoptosis. After cells were treated with PDF (300 μg/mL), VK3 (5 μM), or their combination for 72 h, enzymatic assays were performed for two apoptotic regulators, Csp-3 and Csp-9. The results showed that the PDF/VK3 combination led to activation of Csp-3 and Csp-9 by ~ 2.7 and ~ 3.2 folds, respectively (compared to controls) [Figure 6]. PDF or VK3 alone yet had little effects on Csp-3/9 activities. Since activation of both Csp-3 and Csp-9 has been shown to induce/promote apoptosis,[20],[21] these results are indicative of apoptosis. Therefore, PDF/VK3-induced cell death more likely follows the apoptotic pathway.
Figure 6: Enzymatic activities of caspase-3/9. Cells were treated with PDF (300 μg/mL), Vitamin K3 (5 μM), or their combination for 72 h and subjected to caspase-3/9 enzymatic assays as described in Methods. The spectrophotometric readings were calculated and expressed by fold-increase, compared with the control reading of 1 as indicated by a dotted line. The data are mean ± standard deviation from three separate experiments (*P < 0.05 compared with control)

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  Discussion Top

To explore a more effective treatment for bladder cancer, we investigated the potential anticancer effects of mushroom proteoglucan (PDF), VK3, or their combinations on human bladder cancer T24 cells in vitro. PDF required the relatively high concentrations (≥500 μg/mL) for inducing a significant (P < 0.05) cell viability reduction [Figure 1]a, while VK3 at any given concentrations had little effects [Figure 1]b. However, when PDF and VK3 were combined at their specific concentrations (300 μg/mL PDF and 5 μM VK3), cell viability was drastically reduced by ~ 75% [Figure 2]. Since both PDF (300 μg/mL) and VK3 (5 μM) alone had little effects, such a profound cell viability reduction with their combination may result from “synergism” or a synergistic potentiation of PDF and VK3. In other words, PDF and VK3 appear to work synergistically to induce such a potent anticancer effect. Nevertheless, it was of our interest to also examine if other VK derivatives such as VK1 or VK2 might work just like VK3. Our pilot study showed that VK1 or VK2 by itself had little effects (similar to VK3) but did not yet show any enhanced anticancer effects when they were combined with PDF (data not shown). Thus, specifically VK3, not other VKs, may have such a unique ability to potentiate anticancer effect with PDF.

To have an insight into enhanced anticancer activity of PDF/VK3 combination, cell cycle analysis was performed. Such study indicated that a G1 cell cycle arrest could be the primary cause of the growth cessation, leading to the cell viability reduction. It is plausible that the PDF/VK3 combination could directly interfere with the cell cycle, particularly at the G1-S phase transition. In fact, separate study confirmed that the G1-specific cell cycle regulators (CDK2, cyclin E, and p21Waf1) were indeed modulated with PDF/VK3 treatment [Figure 4], further supporting the resulting G1 cell cycle arrest. Besides this PDF/VK3 combination, the combination of VK3 and VC has also been reported to have a potent cytotoxic effect on several cancer cells at a significantly lower concentration of respective vitamin.[24],[25],[26] Thus, it is conceivable that various agent combinations (including PDF/VK3) may have the better/improved anticancer effect on bladder cancer cells, and such a possibility should be further explored.

In addition, it was tempting to also examine the possible effects of PDF/VK3 combination on epigenetic events as it was insinuated by activation of p21Waf1, which would be regulated by alterations in the acetylation state of histones in the chromatin.[17],[18] We found that HDAC1 was inactivated, leading to hyperacetylation of H3 and H4 [Figure 5] as it has also been shown in elsewhere.[19],[23] This finding confirms that the PDF/VK3 combination is capable of inducing chromatin modifications, subsequently upregulating or activating p21Waf1 [Figure 4]. Therefore, the PDF/VK3 combination seems to modify the chromatin, activate p21Waf1, and then induce a G1 cell cycle arrest. However, there is yet a question that needs to be answered. We may understand that the PDF/VK3 combination can indeed affect HDAC1, H3/H4, p21Waf1, etc., but it is uncertain what would physically/chemically happen when PDF and VK3 were combined – Does any unique chemical reaction take place? Could any (toxic) by-product(s) be released to target HDAC1? Actually, our preliminary study implies that oxidative stress, i.e., generation of reactive oxygen species, could be exerted on the cells when PDF and VK3 were combined. This may deserve further investigations. In the meantime, more studies are required for a much better understanding of exactly how the combination may work.

Finally, whether cell death due to a G1 cell cycle arrest could be feasibly linked to apoptosis was examined. We found that both Csp-3 and Csp-9 were significantly activated with PDF/VK3 treatment [Figure 6]. Csp-3/9 are the specific biochemical parameters known to play a central role in apoptosis and act as the positive or pro-apoptotic regulators – the greater Csp-3/9 activities will more compellingly induce apoptosis.[20],[21] Thus, such activation of Csp-3/9 with the PDF/VK3 combination is indeed indicative of apoptosis, at least in part accounting for the significant cell viability reduction.

After all, this study shows that the PDF/VK3 combination works on bladder cancer T24 cells, but it was not clear whether it would also work on another type of bladder cancer cells or even on any types of cancer cells. In a separate study, we tested the PDF/VK3 combination on another bladder cancer 5637 cells as well as other cancer types such as prostate, kidney, breast, and lung cancer cells. The combination did work, inducing a significant cell viability reduction in all cancer cells tested (data not shown). This finding thus suggests that the PDF/VK3 combination may work commonly on a variety of cancer cells, not in a cancer-specific manner.

Although all these findings seem to be promising, the actual efficacy of PDF/VK3 combination must be adequately assessed in vivo before we draw affirmative conclusion. Particularly, it must be addressed how the effective concentration of PDF/VK3 combination in vitro would be extrapolated to animals or actual patients. In addition, its safety (on normal bladder) and potential side effects (if any) also need to be addressed. Accordingly, the next phase of our study will mainly focus on mice bearing bladder cancer (in vivo)xs to verify/assess the efficacy of PDF/VK3 combination regarding their effective dosages, safety, and side effects.

The present study demonstrates that the combination of PDF and VK3 at specific concentrations appears to be synergistically potentiated to induce the significant (~ 75%) cell viability reduction in T24 cells. Such an enhanced anticancer effect is primarily associated with a G1 cell cycle arrest and chromatin modifications, ultimately leading to apoptosis. Therefore, the combination of PDF and VK3 may provide an unconventional but improved therapeutic modality for, particularly NMIBC case. Further studies are warranted.

Financial support and sponsorship

Financial support for this work was provided by Seize the Ribbon.

Conflicts of interest

There are no conflicts of interest.

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  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6]


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