Transitional cell carcinoma (TCC) of the urinary bladder is the most common cancer in the urinary system. Although the incidence of urinary bladder cancer has continuously increased during the past three decades, there has been little progress in its treatment. Following TUR, tumor recurrence and drug-related toxic side-effects are some of the challenges facing the treatment of bladder cancer. Therefore, the objective of our study was to test a novel approach that was intended to cause significant inhibition of tumor growth with minimal toxicity.
Ki-67 antigen, although often used as a proliferation marker because of its high transcription rate, is beginning to be used as a target in the treatment of various types of cancer 
. The role of curcumin in suppressing the pathways linked to oncogenesis, including cell survival, proliferation, invasion and angiogenesis has already been discussed. Effects on multiple sites such as protein kinase C activation, the alteration of transcriptional factors such as, c-jun/AP-2, NF-κB and p53 by curcumin have been reported 
. However, our studies were aimed at using curcumin as a co-drug since it is now well-established that curcumin can indeed potentiate the effects of anticancer drugs 
. Therefore, the aim of the present study was to evaluate whether we could demonstrate a synergy between the multi-targeted curcumin and Ki-67-7, the anti-Ki-67 siRNA.
In cell culture studies, it was possible to achieve greater than 50% inhibition of tumor cell growth at 20 µM concentration of curcumin. However, we routinely used a sub-maximal dose of 10 µM, to specifically study the combined interaction of curcumin and anti-Ki-67 siRNA. Our data show that although 10 nM of Ki-67-7 significantly inhibited cell proliferation, almost a total inhibition of bladder cancer cell growth was achieved in the combined presence of curcumin (10 µM) (). The advantage of using low concentrations of two separate classes of drugs can be appreciated when we compare our data with those of Zheng et al
, in which, they had to use a ten-fold higher concentration (100 nM) of anti-Ki-67 siRNA to inhibit tumor proliferation only by 45%. Additionally, with regard to curcumin, a lower dose is preferable since increasing its concentration (>50 µM) is also known to be toxic to non-tumor cells 
In the current study, we also attempted to correlate the antiproliferative properties of curcumin and Ki-67-7 on various cellular processes such as apoptosis, cell-cycle progression and Ki-67 expression. Our data show that although curcumin and Ki-67-7 both showed similar degrees of inhibitory effect on cell viability, addition of curcumin to Ki-67-7-treated cells almost completely inhibited cell proliferation with minimal impact on Ki-67 expression (compare and ). This suggested that although curcumin per se had minimal impact on Ki-67, when Ki-67 expression is suppressed by the siRNA, curcumin-mediated processes operate cooperatively to enhance apoptotic cell death in tumor cells. Thus, our data demonstrate for the first time, a synergy between a low concentration of curcumin and an anti-Ki-67 siRNA (Ki-67-7) in the inhibition of bladder cancer cells. The possible molecular targets of the CusiRNA are discussed below.
The initial testing of the interaction between curcumin and Ki-67-7 against bladder cancer cells was measured by MTT assay. Although MTT assay, which determines cell viability, provides the end result, it does not provide information on the events, such as cell cycle arrest and apoptosis that lead to tumor inhibition. An early indicator of apoptosis is the rapid translocation of phosphatidyl serine from the cytoplasmic side of the plasma membrane to the extracellular surface. The loss of asymmetry can be detected by using the binding properties of Annexin V to cell surface. In the present study, a sensitive assay of apoptosis by flow cytometry using Annexin V, clearly demonstrated the synergy between Ki-67-7 and curcumin during the early stages of apoptosis (). Such an observation is reminiscent of the synergy between curcumin and the chemotherapeutic drugs, such as gemcitabine 
and paclitaxel 
, except that siRNAs are far less toxic compared to those drugs.
During cell proliferation, the key cell-cycle events involve transition from one phase to the next; resting (G0), increase in cellular contents (G1), DNA replication (S), chromosome check point and preparation for mitosis (G2) and mitosis (M). Since Ki-67 antigen is involved in DNA replication, an inhibition of this protein was expected to affect the cell cycle phases, specifically at the S phase. As expected, there was a 40% reduction in the number of cells in the S phase when treated with Ki-67-7 and marginal reduction by curcumin. However, in the presence of CusiRNA, a major shift towards growth arrest was observed as indicated by a 2-fold increase in the ratio of G0/G1 to G2/M compared to other treatment groups (). Such a drastic shift in cell cycle phases, in favor of growth arrest and against mitosis (G2/M), was associated with an almost total inhibition of tumor cells (). Kausch et al
have suggested that in the absence of Ki-67, cells that attempt mitosis may be forced into apoptosis. However, our data suggest that the apoptotic process was minimally affected by the inhibition of Ki-67 (caspase-3, ) but profoundly enhanced by the addition of curcumin ().
Although a detailed analysis of the molecular mechanisms underlying the inhibition of tumor cell growth by curcumin and Ki-67-7 is beyond the scope of this study, our data show the following (see for the scheme): Cyclins D1 and E were both inhibited by CusiRNA, thus affecting the cell cycle progression. Reduction in cyclin D1 was also associated with lower pRb-P levels, a downstream activator molecule regulated by cyclin D1. Further, an increase in the level of p27 by CusiRNA, an inhibitor of cyclin E also favored growth arrest. However, the most striking observation of this study is the profound inhibition of both p53 and p21 proteins by CusiRNA (). These two proteins are generally activated by curcumin treatment 
. In an earlier study, Tian et al 
reported that exposure of T-24 cells to curcumin resulted in an increased expression of p53 along with the induction of apoptotic cell death and cell cycle arrest in the G2/M phase. However, unlike in our studies, they had to use a much higher dose (40 µM) of curcumin since the inhibition of tumor growth was low at 10 µM. Since p21 is regulated by p53 
, the inhibition of p53 may explain the absence of p21, another regulator of cyclin E. Nevertheless, the possibility for tumor cells to undergo apoptosis in the absence of p53 and p21, but in the presence of chemotherapeutic drugs has also been reported 
. Taken together, our data suggest that in bladder cancer cells, prior exposure to anti-Ki-67 siRNA, predisposes the tumor cells to curcumin-induced growth arrest and apoptosis by non-p53 and non-p21-dependent pathways.
In summary, our studies show, for the first time, that anti-Ki-67 siRNA, in the combined presence of a low concentration of curcumin, was highly effective against the proliferation of bladder cancer cells. Future studies are required to identify the molecular mechanisms that are involved in the prevention of tumor growth during the combined treatment with anti-Ki-67 siRNA and curcumin. The outcome of our studies could form a new basis for a non-toxic treatment of bladder cancer patients who undergo TUR.