Search for new anti-cancer drugs from natural sources is one of the most important approaches for cancer prevention and treatment. In recent years, more emphasis is laid on Complementary and Alternative [CAM] forms of medicine for the treatment of various cancers, among which herbal medicine is now being explored for cancer therapy . Dietary constituents have chemopreventive and chemotherapeutic potential, in addition to ameliorating the side effects associated with conventional chemotherapy. In this context, a recent approach in cancer therapy advocates the inhibition of the proteolytic activity of 26S proteasome, the multi-enzymatic protease complex in cells. Unlike normal cells, cancer cells have increased proteasomal activity which is essential for their survival and uninhibited proliferation [37–39]. Inhibition of the proteasome results in apoptosis and cancer cell death . Importantly, inhibitors of the 20S proteolytic unit of the proteasome have been shown to induce apoptosis and cell cycle arrest only in neoplastic cells but not in normal cells [39–41]. Therefore, the proteasome has emerged as an attractive molecular target for cancer therapy . A number of synthetic proteasome inhibitors have been described and most of them interfere with the proteolytic activity of the β subunits of the 20S proteasome. These inhibitors which bind the active site either reversibly or irreversibly include peptide aldehydes such as MG-132, non-peptide inhibitors such as lactacystin and epoxomycin and peptide boronates such as bortezomib .
Bortezomib/Velcade/PS-341 is the first-in-line and the only dipeptide boronate proteasome inhibitor to be approved by the FDA in 2003 for the treatment of patients with refractory multiple myeloma. Bortezomib is now being tested in a variety of hematological and solid tumors including non-Hodgkin’s lymphoma, prostate, breast and non-small-cell-lung cancer [44, 45]. In recent years, synthetic polyphenols such as apigenin, epigallocatechin gallate [EGCG], quercitin and myrcetin have been reported to act as proteasome inhibitors and induce cell death in cancer cells .
Drug resistance limits the effectiveness of existing treatment options and is a major challenge faced in current cancer research. Interestingly, it has been shown that lactacystin and bortezomib enhance sensitivity of cancer cells that are resistant to routine chemotherapy [46, 47]. Nevertheless, synthetic proteasome inhibitors are associated with some toxicity. Therefore, proteasome inhibitors from natural food sources with minimal or no toxicity can be potential anticancer agents.
In the present study, we report the anticancer potential of M. koenigii leaf extracts in two human breast carcinoma cell lines. In recent years, dietary polyphenols have attracted lot of attention owing to their anti-tumor activities [48, 49]. One such activity is the inhibition of the proteasome in cancer cells leading to cell death. Recent work from our laboratory  has demonstrated that M. koenigii leaf extract is a rich source of polyphenols. In this study, we found that a hydro-methanolic extract of curry leaves is rich in polyphenol content. Extracts of M. koenigii leaves have been reported to possess various biological activities such as anti-diabetic, anti-oxidative and anti-inflammatory [8–10]. Recently, carbazole alkaloids from M. koenigii have shown anti-cancer activity in leukemia cells [18–20]. However, the underlying mechanism(s) are not reported yet. In the present work, we demonstrate for the first time that the hydro-methanolic extract of curry leaf has proteasome-inhibitory potential and induces cell death in human breast cancer cells.
We found that the methanolic extract of curry leaves significantly decreased cell viability and proliferation of both MCF-7 and MDA-MB-231 breast cancer cells in a dose-dependent manner. This was further supported by the significant reduction in the number of colonies in CLE treated cells compared to vehicle treated cells. Our cell viability and colony formation data shows that CLE altered the growth kinetics of both MCF-7 and MDA-MB-231 cells. Therefore, curry leaves appear to be a promising drug candidate for restricting the growth of breast cancer cells.
In order to assess the stage at which the cell growth was arrested by CLE, we performed cell cycle experiments and observed that there was a clear arrest of cells in the synthetic or S phase. In contrast to its effect on the breast carcinoma cell lines, CLE interestingly, had no effect on the different phases of the cell cycle in the normal fibroblast cell line. Anti-cancer drugs can result either in programmed cell death/apoptosis or necrosis. In order to identify the probable cell death pathway involved, we used Annexin V binding to test if the cell death occurred through apoptosis or necrosis. Indeed it was found that in both the breast cancer cell lines CLE induced apoptotic cell death.
We tested next, whether the anti-cancerous effect of the CLE was due its potential of inhibiting the proteolytic activity of the protein degrading machine present in eukaryotic cells – the 26S proteasome, which is now considered to be a novel approach for cancer therapy. We observed that CLE inhibited the purified 20S proteasome enzyme. Furthermore, it significantly inhibited all the three enzymatic activities associated with the 26S proteasome in living cells in a dose-dependent manner. To further confirm the findings in live cells, cell extracts were prepared from both breast cancer cells and tested for the potential of the CLE in inhibiting the cellular proteasome. Similar to our findings in intact cells we found that CLE inhibited the 26S proteasome in cell extracts also in a dose-dependent manner. To test whether or not the inhibitory effect of the CLE on the proteasome activity was specific to cancer cells, we tested its effects in WI-38, a normal lung fibroblast cell line. Interestingly, CLE had no effect on the chymotrypsin–like activity of the 26S proteasome in live WI-38 cells. Hence, data from our proteasome-inhibition experiments suggests that the CLE could inhibit the cellular proteasome leading to cell death in cancer cells but not normal cells. Our data is in accordance with earlier reports that proteasome inhibitors selectively inhibited proteasome activity only in neoplastic cells [39, 50, 51].