Alternative Therapeutic Approach to Renal-Cell Carcinoma: Induction of Apoptosis with Combination of Vitamin K 3 and D-fraction

Abstract

Purpose:

Because of a dismal prognosis for advanced renal-cell carcinoma (RCC), an alternative therapeutic approach, using vitamin K₃ (VK₃) and D-fraction (DF) was investigated. VK₃ is a synthetic VK derivative and DF is a bioactive mushroom extract, and they have been shown to have antitumor activity. We examined if the combination of VK₃ and DF would exhibit the improved anticancer effect on RCC in vitro.

Materials and Methods:

Human RCC, ACHN cell line, were treated with varying concentrations of VK₃, DF, or a combination of the two. Cell viability was assessed at 72 hours by MTT assay. To explore the possible anticancer mechanism, studies on cell cycle, chromatin modifications, and apoptosis were conducted.

Results:

VK₃ alone led to a ∼20% reduction in cell viability at 4 μM, while DF alone induced a 20% to 45% viability reduction at ≥500 μg/mL. A combination of VK₃ (4 μM) and DF (300 μg/mL) led to a drastic >90% viability reduction, however. Cell cycle analysis indicated that VK₃/DF treatment induced a G₁ cell cycle arrest, accompanied by the up-regulation of p21^WAF1 and p27^Kip1. Histone deacetylase (HDAC) was also significantly (∼60%) inactivated, indicating chromatin modifications. In addition, Western blot analysis revealed that the up-regulation of Bax and activation of poly-(ADP-ribose)-polymerase (PARP) were seen in VK₃/DF-treated cells, indicating induction of apoptosis.

Conclusions:

The combination of VK₃ and DF can lead to a profound reduction in ACHN cell viability, through a p21^WAF1-mediated G₁ cell cycle arrest, and ultimately induces apoptosis. Therefore, the combination of VK₃/DF may have clinical implications as an alternative, improved therapeutic modality for advanced RCC.

Introduction

Renal-cell carcinoma (RCC) is the third most common genitourinary tumor in the United States,¹ and nearly 30% of patients with RCC will present with metastatic disease.^2,3 Standard treatment for nonmetastatic RCC is complete resection of the tumor by either a radical or partial nephrectomy^4,5; however, 20% to 30% of these patients will progress to metastatic disease. While a majority of these patients in whom metastsis develops after surgery presented with larger tumors necessitating radical nephrectomies, a recent study suggested that distant metastasis developed in 4% of patients who had positive surgical margins after nephron-sparing surgery, commonly performed laparoscopically or robotically.⁶ With a 5-year survival rate of <10% for metastatic RCC,⁷ these patients are left with a dismal prognosis, making RCC one of the most fatal genitourinary cancers.

Unfortunately, conventional therapeutic modalities for metastatic RCC, including cytoreductive surgery, radiotherapy, chemotherapy, or immunotherapy, were not very effective.^3,8 Newer, specifically targeted treatment options for distant metastasis, including tyrosine kinase inhibitors (sunitinib, etc.), mammalian target of rapamycin inhibitors (temsirolimus, etc.), and monoclonal antibodies (G250, etc.), are promising, but the survival results are still poor.⁹ These reasons thus prompted us to explore a novel treatment for patients with metastatic RCC.

We have been working with vitamins and natural agents/substances as an alternative approach to such metastatic RCC cases. Particularly, we were interested in vitamin K₃ (menadione; VK₃) as a potential therapeutic agent. VK₃ is a synthetic derivative of naturally occurring fat-soluble vitamin K (VK)¹⁰ and has been shown to have an antitumor effect on several cancer cells.^11
–13 Because VK₃ requires a relatively high dose to be effective, ¹¹ combinations of VK₃ and other agents have been postulated to improve its efficacy at a lower dose.

At the same time, we have also been studying the bioactive extract from maitake (Grifola frondosa), known as D-fraction (DF). DF has been extensively studied for the past 30 years and has been shown to have immunomodulatory and antitumor activities.^14
–16 For instance, antitumor activity of DF has been demonstrated in tumor-bearing mice through activation of various immune effectors.^14,15 In addition, induction of apoptosis by DF was also reported in breast cancer cells.¹⁶ DF thus appears to be an interesting and promising natural agent that could be used in cancer treatment.

The purpose of this study was to investigate whether a combination of VK₃ and DF would exhibit the improved and enhanced anticancer effect on RCC in vitro, as an alternative therapeutic approach. These studies were performed focusing mainly on cell viability, cell cycle, chromatin modifications, and apoptosis.

Materials and Methods

Cell culture

The human renal carcinoma ACHN cells (with aggressive grade IV property) were maintained in RPMI 1640 medium containing 10% fetal bovine serum, penicillin (100 units/mL), and streptomycin (100 μg/mL). For experiments, ACHN cells were seeded at the initial cell density of 2×10⁵ cells/mL in six-well plates or T-75 flasks and cultured with varying concentrations of VK₃, DF, or their combinations. Cell viability was then assessed at 72 hours by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) assay.

Cell viability assay (MTT assay)

Cell viability was determined by MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) assay following the protocol of the vendor (Sigma-Aldrich, St. Louis, MO). Briefly, at the harvest time, MTT reagent (0.5 mg/mL) was added to each well in the six-well plate, followed by 3-hour incubation at 37°C. After removing MTT reagent, dimethyl sulfoxide was added to each well and absorbance of formazan solution was read on a microplate reader. Cell viability was then expressed as percentage (%) of viable cells relative to the control reading (100%).

Cell cycle analysis

A BD FACscan flow cytometer (Franklin Lakes, NJ), equipped with a double discrimination module, was used for cell cycle analysis. Approximately 1×10⁶ cells were resuspended in 500 μL of propidium iodide solution and incubated at room temperature for 1 hour. Ten thousand nuclei were analyzed for each sample, quantified in cell cycle compartments, and estimated as cell cycle phase fractions.

Histone deacetylase (HDAC) assay

HDAC activity was measured using the Epigenase HDAC Assay Kit following the protocol of the manufacturer (Epigentek, Farmingdale, NY). Briefly, 10 μg of nuclear extracts from each sample were added to the coated microplate wells. After 90-minute incubation at 37°C, all wells in the plate were treated with 1st antibody for 60 minutes, followed by 30-minute incubation with 2nd antibody. The plate was then treated with reaction solution for 10 minutes, and the reaction was terminated with stop solution. Absorbance of samples was read on a microplate reader, and HDAC activity was calculated and expressed by the % relative to controls (100%).

Western blot analysis

The detailed procedures are described elsewhere.¹⁷ An equal amount of cell lysates (7 μg), obtained from control and agent-treated cells, was first subjected to 10% SDS gel electrophoresis and transferred to a nitrocellulose membrane (blot). The blot was incubated for 90 minutes with the primary antibodies against p21^WAF1, p27^Kip1, Bax, or poly-(ADP-ribose)-polymerase (PARP) (Santa Cruz Biotechnology, Santa Cruz, CA), followed by 30-minute incubation with the secondary antibody conjugates. The specific immunoreactive protein bands were then detected by chemiluminescence following the protocol of the manufacturer (KPL, Gaithersburg, MD).

Statistical analysis

All data were presented as mean±standard deviation, and statistical differences between groups were assessed with either one-way analysis of variance or the unpaired Student t test. Values of P<0.05 were considered to indicate statistical significance.

Results

Effects of VK₃, DF, or their combinations on ACHN cell growth

ACHN cells were cultured separately with varying concentrations of VK₃ (0–4 μM) or DF (0–700 μg/mL). After 72 hours, MTT assay showed that VK₃ alone had little effects but led to a ∼20% reduction in cell viability at 4 μM (Fig. 1A), while DF induced a 20% to 45% viability reduction at ≥500 μg/mL (Fig. 1B). Whether the combination of VK₃ and DF may potentiate the anticancer effect was also examined. Such study showed that a profound cell viability reduction, from severe (∼90%) cell death, was attained with the specific combination of 4 μM VK₃ and 300 μg/mL DF (Fig. 2).

FIG. 1.

(A) Effects of vitamin K₃ (VK₃) on cell viability of ACHN cells. After cells were cultured with varying concentrations of VK₃ (0–4 μM) for 72 hours, cell viability was assessed by MTT assay. (B) Effects of D-fraction (DF) on cell viability. After cells were treated with DF (0–700 μg/mL) for 72 hours, cell viability was then determined. Cell viability was expressed by the % of viable cell number relative to controls (100%). The data are mean±standard deviation from three separate experiments (*P<0.05 compared with respective controls).

FIG. 2.

Effects of combination of vitamin K₃ (VK₃) and D-fraction (DF) on cell viability. After cells were treated with VK₃ (4 μM), DF (300 μg/mL), or their combination for 72 hours, cell viability was assessed by MTT assay and expressed by the % relative to controls. The data are mean±standard deviation from three separate experiments (*P<0.05 compared with controls).

Effects of VK₃/DF combination on cell cycle

To explore how such VK₃/DF combination would induce a viability reduction or cell death, cell cycle analysis was performed. Cells exposed to VK₃/DF combination for 6 hours showed a 42% increase in the G₁-phase cell number with a 60% decrease in the S-phase cells (Fig. 3). This accumulation of cells in the G₁ phase, from a blockade of the cell cycle transition from the G₁ to the S phase, is known as a G₁ cell cycle arrest.¹⁸ Thus, VK₃/DF combination appears to disrupt the cell cycle at a G₁ checkpoint.

FIG. 3.

Cell cycle analysis. Cells treated with or without vitamin K₃ (VK₃) (4 μM)/ D-fraction (DF) (300 μg/mL) combination for 6 hours were subjected to cell cycle analysis, and the % of cell distribution was plotted against the respective cell cycle phase. The data are mean±standard deviation from three separate experiments (*P<0.01 compared with controls).

Effects of VK₃/DF combination on G₁ cell cycle regulators

VK₃/DF-induced G₁ cell cycle arrest was further confirmed by analyzing the specific cell cycle regulators for the G₁-S phase transition. Western blots revealed that compared with controls, the expressions of two G₁ cyclin-dependent kinase inhibitors, p21^WAF1 and p27^Kip1, were significantly enhanced or up-regulated with VK₃/DF treatment for 72 hours (Fig. 4). Because this up-regulation of p21^WAF1 and p27^Kip1 is indicative of a G₁ cell cycle arrest,¹⁸ the VK₃/DF combination appears to induce a G₁ arrest, leading to a growth cessation and consequently resulting in the cell viability reduction.

FIG. 4.

Analysis of G₁ cell cycle regulators. Expressions of p21^WAF1 and p27^Kip1 in cells treated with vitamin K₃ (VK₃) (4 μM)/D-fraction (DF) (300 μg/mL) combination for 72 hours were analyzed on Western blots. Autoradiographs of p21^WAF1 and p27^Kip1 in control and VK₃/DF-treated cells are shown for comparison.

Effects of VK₃/DF combination on HDAC activity

Because p21^WAF1 is known to be regulated by chromatin modifications from alterations in the acetylation state of histones,^19,20 such a possibility was tested on HDAC, one of the primary regulators of histone acetylation.²¹ Cells were treated with VK₃ (4 μM), DF (300 μg/mL), or their combination for 24 hours. Assays showed that VK₃ or DF alone had little effects whereas the VK₃/DF combination led to a ∼60% reduction in HDAC activity (Table 1). Thus, these results suggest that the VK₃/DF combination may significantly inactivate HDAC, inducing hyperacetylation of specific histones¹⁹ that could then up-regulate p21^WAF1 as shown in Fig. 4.

Table 1.

Effects of Vitamin K₃, D-Fraction, or Their Combination on HDAC Activity

Conditions	HDAC activity (%) at 24 hours^a
Control	100
+ VK₃ (4 μM)	92±2.6 (∼8%↓)^b
+ DF (300 μg/mL)	97±2.1 (∼3%↓)
+ VK₃ (4 μM) / DF (300 μg/mL)	39±3.2 (∼61%↓)

Mean±standard deviation of three separate experiments (control 100%=0.91 OD/min/mg).

Values in parentheses are the percent (%) of reduction in HDAC activity relative to controls.

HDAC=histone deacetylase; VK₃=vitamin K₃; DF=D-fraction; OD=absorbance reading at 450 nm.

Effects of VK₃/DF combination on apoptosis regulators

Because it was of interest to also explore how the VK₃/DF combination would induce cell death, the possible role of apoptosis was then investigated. Cells treated with the VK₃/DF combination for 12 hours were analyzed for expressions of two key apoptosis regulators on Western blots. VK₃/DF treatment led to the up-regulation of pro-apoptotic Bax and activation of pro-apoptotic PARP (Fig. 5). Because the up-regulation/activation of Bax and PARP indicates induction of apoptosis,^22,23 it is plausible that VK₃/DF-induced cell death may follow apoptosis.

FIG. 5.

Effects of vitamin K₃/D-fraction (VK₃/DF) on apoptosis regulators. Expressions of Bax and poly-(ADP-ribose)-polymerase (PARP) in cells exposed to VK₃ (4 μM)/DF (300 μg/mL) combination for 12 hours were analyzed on Western blots. Autoradiographs of Bax and PARP in control and VK₃/DF-treated cells are shown.

Discussion

To explore an alternative, minimally invasive, and more effective treatment for advanced RCC, we investigated the potential anticancer effect of VK₃ and DF on human renal carcinoma ACHN cells. VK₃ by itself had some effect with a ∼20% cell viability reduction, while DF needed relatively high concentrations (≥700 μg/mL) to be effective. When two agents with varying concentrations were combined, however, a specific combination of 4 μM VK₃ and 300 μg/mL DF led to a drastic ∼90% reduction in cell viability, due to severe cell death (Fig. 2). This finding was somewhat extraordinary and prompted us to explore the cytotoxic mechanism of the VK₃/DF combination.

To obtain a better understanding of the VK₃/DF cytotoxic mechanism, cell cycle analysis was performed. The results indicated that a G₁ cell cycle arrest could be the primary cause of a growth cessation (Fig. 3), resulting in a cell viability reduction. A G₁ cell cycle arrest was also confirmed by the up-regulation of both p21^WAF1 and p27^Kip1 (Fig. 4), which were the inhibitors of the G₁-specific cell cycle regulators.¹⁸ These findings suggest that a VK₃/DF combination could directly interfere with the cell cycle, particularly at the G₁-S phase transition.

It is also plausible that a G₁ cell cycle arrest could be a prerequisite for cell death induced by the VK₃/DF combination. In particular, p21^WAF1 has been shown to be associated with a G₁ cell cycle arrest³¹ and its up-regulation (inducing a G₁ arrest) is also known to result from histone modifications in the chromatin.¹⁹ Our study revealed that HDAC was significantly (∼60%) inhibited by the VK₃/DF combination (Table 1), presumably inducing hyperacetylation of histones that would then up-regulate p21^WAF1. This finding thus suggests that the VK₃/DF combination may induce chromatin modifications. The actual acetylation state of specific histones H3 and H4 with other related factors, however, needs to be further examined for confirming such key histone modifications. This study is under way in our laboratory.

Moreover, it was our rational attempt to examine whether the VK₃/DF-induced cell death might be associated with apoptosis because it could provide us with valuable information. We then found that two pro-apoptotic regulators, Bax and PARP, were clearly up-regulated with the VK₃/DF combination (Fig. 5), indicating induction of apoptosis.^22,23 Thus, the VK₃/DF combination appears to ultimately induce apoptotic cell death, accounting for the profound cell viability reduction.

Finally, it is worth discussing the safety profile of DF because of its possible clinical implications. The United States Food and Drug Administration has exempted DF from a phase I study of toxicology and approved it for the Investigational New Drug application for a phase II pilot study on advanced cancer patients.²⁴ Therefore, the safety of DF has been granted, and it is safe to be used in both normal subjects and a variety of patients. Regarding VK₃, it has been studied in several cancer cells,^11
–13 and we believe that the effective concentrations of VK₃ (when combined with DF) would be more likely safe and nontoxic. We are going to perform a toxicology test on VK₃ for confirmation using animals in our next study, however.

The findings in this study are interesting and encouraging. It is possible that the combination of VK₃ and DF could be used as an adjuvant therapy to help reduce the incidence of potential recurrence after surgical treatment of RCC. However, preclinical studies (using animals) are required, however, for assessing the safety and true efficacy of the VK₃/DF combination before clinical trials. Accordingly, the next phase of our study will primarily focus on the actual effect of the VK₃/DF combination on RCC-bearing animals (athymic nude mice).

Conclusions

The present study demonstrates that the combination of VK₃ and DF is highly cytotoxic, inducing a ∼90% cell death in ACHN cells. Such cell death is preceded with a p21-dependent G₁ cell cycle arrest presumably through histone modifications. This cell cycle arrest will ultimately lead to apoptosis. Therefore, the VK₃/DF combination appears to be promising and may have clinical implications as an alternative and effective therapeutic modality for advanced RCC.

Footnotes

Disclosure Statement

No competing financial interests exist.

Abbreviations Used

References

Jemal

, Siegel

, Xu

, Ward

. Cancer statistics, 2010. CA Cancer J Clin, 2010; 60:277–300.

Ries

LAG

, Eisner

, Kosary

, et al (eds). SEER Cancer Statistics Review, 1975–2001. National Cancer Institute, Bethesda, MD, 2004.

Jacobsohn

, Wood

. Adjuvant therapy for renal cell carcinoma. Semin Oncol, 2006; 33:576–582.

Cohen

, McGovern

. Renal-cell carcinoma. N Engl J Med, 2005; 353:2477–2490.

Kunkle

, Uzzo

. Cryoablation or radiofrequency ablation of the small renal mass: A meta-analysis. Cancer, 2008; 113:2671–2680.

Kwon

, Carver

, Snyder

, Russo

. Impact of positive surgical margins in patients undergoing partial nephrectomy for renal cortical tumours. BJU Int, 2007; 99:286–289.

Motzer

, Russo

. Systemic therapy for renal cell carcinoma. J Urol, 2000; 163:408–417.

Glaspy

. Therapeutic options in the management of renal cell carcinoma. Semin Oncol, 2002; 29(suppl 7):41–46.

Motzer

, Agarwal

, Beard

, et al. NCCN clinical practice guidelines in oncology: Kidney cancer. J Natl Compr Canc Netw, 2009; 7:618–630.

10.

Shearer

. Role of vitamin K and Gla proteins in the pathophysiology of osteoporosis and vascular calcification. Curr Opin Clin Nutr Metab Care, 2000; 3:433–438.

11.

, Liao

, Chang

. Comparison of antitumor activity of vitamins K1, K2 and K3 on human tumor cells by two (MTT and SRB) cell viability assays. Life Sci, 1993; 52:1797–1804.

12.

Lamson

, Plaza

. The anticancer effects of vitamin K. Altern Med Rev, 2003; 8:303–318.

13.

Ogawa

, Nakai

, Deguchi

, et al. Vitamins K2, K3 and K5 exert antitumor effects on established colorectal cancer in mice by inducing apoptotic death of tumor cells. Int J Oncol, 2007; 31:323–331.

14.

Hishida

, Nanba

, Kuroda

. Antitumor activity exhibited by orally administered extract from fruit body of Grifola frondosa (maitake). Chem Pharm Bull (Tokyo), 1988; 36:1819–1827.

15.

Adachi

, Nanba

, Kuroda

. Potentiation of host-mediated antitumor activity in mice by β-glucan obtained from Grifola frondosa (maitake). Chem Pharm Bull (Tokyo), 1987; 35:262–270.

16.

Soares

, Meireles

, Rocha

, et al. Maitake (D fraction) mushroom extract induces apoptosis in breast cancer cells by BAK-1 gene activation. J Med Food, 2011; 14:563–572.

17.

Mordente

, Konno

, Chen

, et al. The effects of brefeldin A (BFA) on cell cycle progression involving the modulation of the retinoblastoma protein (pRB) in PC-3 prostate cancer cells. J Urol, 1998; 159:275–279.

18.

Sherr

, Roberts

. CDK inhibitors: Positive and negative regulators of G₁-phase progression. Genes Dev, 1999; 13:1501–1512.

19.

Richon

, Sandhoff

, Rifkind

, Marks

. Histone deacetylase inhibitor selectively induces p21^WAF1 expression and gene-associated histone acetylation. Proc Natl Acad Sci U S A, 2000; 97:10014–10019.

20.

Villar-Garea

, Esteller

. Histone deacetylase inhibitors: Understanding a new wave of anticancer agents. Int J Cancer, 2004; 112:171–178.

21.

de Ruijter

, van Gennip

, Caron

, et al. Histone deacetylases (HDACs): characterization of the classical HDAC family. Biochem J, 2003; 370:737–749.

22.

Yip

, Reed

. Bcl-2 family proteins and cancer. Oncogene, 2008; 27:6398–6406.

23.

Soldani

, Scovassi

. Poly(ADP-ribose) polymerase-1 cleavage during apoptosis: An update. Apoptosis, 2002; 7:321–328.

24.

Maitake Products Inc. D-fraction obtained IND for clinical study. Corporate Publication, Paramus, NJ, 1998.