The suppressive effect of Rho kinase inhibitor, Y-27632, on oncogenic Ras/RhoA induced invasion/migration of human bladder cancer TSGH cells

Abstract

Urothelial cell carcinoma is the most common type of malignancy found in long-term dialysis patients and kidney transplant recipients in Taiwan. Surgical specimens of tumorous and non-tumorous bladder tissues were collected from 12 patients with bladder cancer. Increased expressions of Ras, RhoA, Akt, PI-3K were demonstrated in the tumors as compared to adjacent control tissues. To understand the impact of Ras over-expression on bladder cancer progression, human bladder cancer TSGH 8301 cells were transfected with Ras DNA. The Ras-transfected cells were then treated with either a PI-3K inhibitor (wortmannin) or Rho kinase inhibitor (Y-27632) and the expressions of Ras, PI-3K, Akt, NF-κB, and RhoA were analyzed. Fluorescent phalloidin staining demonstrated more intense F-actin staining in the Ras over-expressed cells than in the control cells, and the intensity of F-actin was inhibited by Y-27632. A gelatin zymography study demonstrated that the MMP-2 and MMP-9 expressions of the Ras-transfected cells were enhanced, and Y-27632 treatment reduced the levels of MMP-2 and MMP-9. Similarly, a wound healing assay revealed that the ability of cell migration was markedly increased by Ras transfection and the healing rate after treatment of Y-27632 was delayed. Our results provide evidence that Ras-induced RhoA and NF-κB activation was involved in the invasion/migration of bladder cancer. Through Ras and/or RhoA inhibition, there might be an opportunity for new therapeutic interventions in bladder cancer.

Introduction

Bladder carcinoma is the most common malignancy of the urinary tract, and 90% of bladder carcinomas are urothelial cell carcinomas (UCCs). At the initial diagnosis of bladder cancer, 70% of cases are diagnosed as non-muscle-invasive disease and 30% as muscle-invasive disease. The 2004 WHO grading system classifies papillary urothelial carcinoma into only two grades: (1) low-grade papillary urothelial carcinoma exhibiting an overall orderly appearance, but with minimal variability in architecture and/or cytologic features, which are easily recognizable at scanning magnification. (2) High-grade papillary urothelial carcinomas which are characterized by a disorderly appearance from marked architectural and cytologic abnormalities, recognizable at low magnification [1]. The risk factors for bladder cancer include tobacco smoking and occupational exposure which are the most well-established factors [2]. The substances most commonly involved in occupational exposure are polycyclic aromatic hydrocarbons and arylamines. Professions in which this exposure occurs include those that use dyes, rubbers, textiles, paints, leathers and chemicals [3]. Other risk factors include elevated arsenic levels in drinking water [4], phenacetin, radiation therapy, dietary factors and chronic urinary tract infections. The cancer prevalence rates in patients with end-stage renal disease reported by the United States Renal Data System (USRDS, 2005) involves the digestive system (13.6%), reproductive system (9%), kidneys (4.2%), skeletal cancer (2.7%), skin cancer (2.4%), lymphoid tissue (2.1%) and respiratory system (1.7%) [5]. However, UCC is the most common malignancy found in long-term dialysis patients in Taiwan, and chronic tubulointerstitial nephritis is the most likely underlying renal disease [6]. UCC is also the most common type of cancer in kidney transplant recipients in Taiwan, with an incidence of 4.1% [7] and a high percentage of 43.6 among all post-transplant cancers [8]. The incidence of UCC in the general population was 2.76% according to the 2005 annual report of the Taiwan cancer registry. The risk factors predisposing to UCC in Taiwan include being of older age, underground water intake, being female, compound analgesics use as well as Chinese herbs use [6], [7], [9], [10]. Therefore, in regard to developing chronic tubulointerstitial nephropathy and UCC, chronic usage of analgesics and Chinese herbs are particular and important risk factors in Taiwan. Chronic usage of analgesics is associated with those of a lower social-economic status, and the use of aristolochic acid was not banned until 2003 in Taiwan.

Although bladder cancer is a chemosensitive neoplasm, metastatic disease is related with a poor prognosis and short-term survival. For two decades, the treatment of choice for metastatic bladder cancer has been cisplatin-based chemotherapy. More recently, non-platinum regimes have been tested, such as taxanes and gemcitabine, which are considered attractive alternatives. However, owing to treatment-related toxicities and short-response durations, identification of more molecular prognostic factors and application of targeted therapies have been sought with considerable interest.

Ras oncogenes are considered to play a key role in the carcinogenesis and progression in several cancers. Activation of Ras proteins has been reported to induce the constitutive activation of downstream kinase cascades, which results in continuous mitogenic signaling and transformation of immortalized cells in human bladder cancer [11]. Additionally, Rho is important for the ability of tumor cells to metastasize [12]. Kamai et al. demonstrated that RhoA, RhoC, and Rho kinase (ROCK), but not RhoB protein expressions were greater in bladder tumors [13]. Although both Ras and RhoA have been reported to be associated with bladder tumors, to the best of our knowledge, a relationship between Ras, RhoA signaling and the invasion/migration process of human bladder cancer has not been described. Thus, the aim of this study was to clarify the association between Ras and RhoA and their impact on the invasion/migration of human bladder cancer.