Cell cycle arrest and autoschizis in a human bladder carcinoma cell line following Vitamin C and Vitamin K₃ treatment

Abstract

Exponentially growing cultures of human bladder tumor cells (T24) were treated with Vitamin C (VC) alone, Vitamin K₃ (VK₃) alone, or with a VC:VK₃ combination for 1, 2, or 4 hr. Flow cytometry of T24 cells exposed to the vitamins for 1 h revealed a growth arrested population and a population undergoing cell death. Cells in G₁ during vitamin treatment arrested in G₁ while those in S phase progressed through S phase and arrested in G₂/M. DNA synthesis decreased to 14 to 21% of control levels which agreed with the percent of cells in S phase during treatment. Annexin V labeling demonstrated the majority of the cells died by autoschizis, but necrosis and apoptosis also were observed. Catalase treatment abrogated both cell cycle arrest and cell death which implicated hydrogen peroxide (H₂O₂) in these processes. Redox cycling of VC and VK₃ increased H₂O₂ production and decreased cellular thiol levels and DNA content, while increasing intracellular Ca²⁺ levels and lipid peroxidation. Feulgen staining of treated cells revealed a time-dependent decrease in tumor cell DNA, while electrophoresis revealed a spread pattern. These results suggest that Ca²⁺ disregulation activates at least one DNase which degrades tumor cell DNA and induces tumor cell death.

Introduction

The bladder is the most common site of neoplasm in the urinary system and accounts for approximately 29% of all urologic tumors [1]. In 2002, it is estimated that 56,500 new bladder cases would be diagnosed and 12,600 deaths would result from bladder cancer in the United States [2]. Because of the multifocal nature of these tumors, instillation therapy (flooding of the bladder with a solution of chemotherapeutic or immunostimulating agents) is a common practice. However, instillation therapy of bladder carcinomas often requires repeated application of antitumor agents over a period of several years. Unfortunately because of the dose-limiting toxicity of chemotherapeutic agents, resistance to neoplastic agents, and recurrence of the cancer, a cure has remained elusive for many cancer patients [3]. Therefore, there is a substantial need for new therapeutic options with favorable pharmacokinetics and pharmacodynamic parameters as well as a selective mechanism of action.

Due to their low systemic toxicity, several vitamins have been evaluated for their abilities to prevent or treat cancer [4]. Vitamins A, B₆, C, E, and K₃ have all demonstrated activity in the prevention or treatment of bladder cancer [5], [6], [7], [8]. In addition, Lamm et al.[4] have demonstrated that oral administration of megadoses of Vitamins A, B₆, C, and E in conjunction with zinc markedly reduced tumor recurrence in patients with transitional cell carcinoma. This vitamin treatment was nontoxic and produced a greater reduction in the rate of tumor recurrence than BCG immunotherapy which is the gold standard for the treatment of superficial bladder cancer. To date, the mechanism(s) providing this protection have not been elucidated.

Vitamin C and Vitamin K₃ also exhibit antitumor activity against bladder cancer [8], [9]. When Vitamin C and Vitamin K₃ are combined in a VC:VK₃ ratio of 100:1, the combination exhibits tumor-specific antitumor activity against human breast, oral epidermoid, and endometrial tumor cell lines at doses which are 10–50 times lower than when either vitamin is administered alone [10]. Additional studies using hepatoma-bearing mice as a model have shown that the VC:VK₃ combination alone exhibits antitumor activity [11]. In addition, the VC:VK₃ combination has been shown to be an effective chemosensitizer and radiosensitizer that induces little systemic or major organ pathology [11], [12]. Studies conducted in our laboratory demonstrate that the VC:VK₃ combination exhibits synergistic antitumor activity against a panel of human urologic tumor cell lines and against human prostate cancer cells (DU145) which have been implanted into nude mice [13]. The results of more recent studies with the T24 human bladder cancer cell line [14], [15], [16], [17], demonstrated that Vitamin C and Vitamin K₃ treatment induced cell death by autoschizis.

In the current study, the antitumor activity of VC, VK₃, and the VC:VK₃ combination against the T24 human bladder cancer cell line has been determined following a continuous or a 1-hr pulse exposure to the vitamins. The 1-hr pulse vitamin exposure has been performed in an attempt to simulate exposure of bladder cancer cells to the vitamins during instillation therapy (the chemotherapeutic agent typically resides in the bladder for 1 hr or less). Flow cytometry, histochemistry, and bioassays have been employed to elucidate vitamin-induced changes in cell cycle, cell viability, cellular DNA content and status and cellular thiol content.