MTT Survival Assays Research Articles

(S)‐HDAC‐42, A New HDAC Inhibitor in Ovarian Cancer Therapy

Although the current choice for ovarian cancer treatment is still cisplatin, the resistance to cisplatin therapy remains a critical obstacle in the management of refractory ovarian cancer. In the search for new therapies, histone deacetylases (HDACs) have been considered one of the most promising targets. HDAC inhibitors, such as trichostatin A (TSA) and suberoylanilide hydroxamic acid (SAHA), have been reported to be effective against ovarian cancer cells in vitro and in vivo.. We have developed a new class of HDAC inhibitors by linking short-chain fatty acids with Zn2+-chelating motifs. Further structure-based optimization of these novel hydroxamate-tethered phenylbutyrate derivatives (HTPB) culminated in the generation of (S)-HDAC42, which, in this study, we show is a potent anti-cancer agent for ovarian cancer in vitro and in vivo. The treatment of both cisplatin-sensitive and -resistant ovarian cancer cell lines with (S)-HDAC42 restored histone acetylation and suppressed cell growth with IC50 values of about 1μM as determined by the MTT survival assay after 2 days treatment. Additionally, (S)-HDAC42 caused the accumulation of p21 and p27, down-regulation of Cdc2 and cyclin B1, and induced cell cycle arrest at G2/M phase. In the mean time, (S)-HDAC42 also induced cell differentiation and apoptosis. Furthermore, (S)-HDAC42 treatment of xenograft-bearing nude mice significantly inhibited tumor growth of the cisplatin-resistant ovarian carcinoma cell line CP70 compared to vehicle treatment. Moreover, the combination of (S)-HDAC42 and cisplatin treatments induced an enhanced suppression of tumor growth. In conclusion, these findings indicate that the novel HDAC inhibitor, (S)-HDAC42, is a promising agent for the treatment of ovarian cancer.

Investigation of multidrug resistance in cultured human renal cell carcinoma cells by 31P-NMR spectroscopy and treatment survival assays

KTCTL-26 and KTCTL-2 are renal cell carcinoma (RCC) lines with high and low expression of P-170 glycoprotein, respectively. Inherent differences between the two cell lines in terms of phosphate metabolites and growth characteristics in culture were examined for possible association with multidrug resistance (MDR). Differences in response to drug treatment were investigated for 40 h incubations with various doses of vinblastine (VBL) alone or as cotreatments with various concentrations of the calcium antagonist diltiazem (DIL) and/or interferon-alpha (IFN-alpha). Treatment effects were quantitated using the MTT survival assay and 31P magnetic resonance spectroscopy (MRS) to determine phosphate metabolite profiles in intact cells. KTCTL-2 and KTCTL-26 cells exhibited significant inherent differences in phosphocholine, glycerophosphocholine, glycerophosphoethanolamine, and phosphocreatine levels. KTCTL-26 cells were more sensitive than KTCTL-2 to 0.011 mircroM VBL alone (87% vs. 102% survival) or to 0.011 microM BL + 10 microM DIL (55% vs. 80% survival). The latter treatment resulted in a significant decrease in the ratio of phosphocholine to glycerophosphocholine in KTCTL-26 cells but no significant changes in phosphate metabolites in KTCTL-2 cells. Metabolomic 31P MRS detects different metabolite profiles for RCC cell lines with different MDR phenotypes and may be useful for noninvasive characterization of tumors in a clinical setting.

Avicins: A Novel Class of Anti-Myeloma Agents.

Multiple myeloma (MM) remains an incurable neoplasia, despite recent development of several novel therapies. As part of our efforts to identify new compounds with anti-MM activity, we evaluated the class of avicins, which are triterpenoid saponins that have been shown to induce apoptosis of neoplastic cells by affecting mitochondrial function independently of membrane-bound death receptors. Because we have previously shown that mitochondria constitute key regulators of MM cell responsiveness to diverse anti-tumor agents, (e.g. the proteasome inhibitor bortezomib), we evaluated the in vitro anti-MM effects of this class of compounds. Our vitro drug-sensitivity studies showed that Avicin D and Avicin G, the main members of this class of compounds, are active against a broad panel of MM cell lines and primary tumor cells, including tumor cells resistant to conventional (e.g. dexamethasone, alkylating agents, anthracyclines) or novel (e.g. thalidomide, immunomodulatory thalidomide derivatives, proteasome inhibitor PS-341[bortezomib], Apo2L/TRAIL) anti-MM agents. Using MTT survival assays, we confirmed that the IC50 values for both Avicins were highly concordant and were less than 250 nM for the overwhelming majority of MM cell lines tested. Importantly, this potent in vitro anti-MM activity was triggered by concentrations of Avicins which had minimal, if any, effect on the viability of normal hematopoietic cells or bone marrow stromal cells. Furthermore these IC50 values were comparable with the in vitro activity of this agent among the most Avicin-sensitive tumor models that have been previously tested. This potent anti-MM effect was not inhibited by transfection of MM cells with construct for constitutively active Akt. Although cytokine- or cell adhesion-mediated interactions of the local bone marrow (BM) microenvironment (e.g. BM stromal cells) protects MM cells from conventional therapies (e.g. dexamethasone or cytotoxic chemotherapy), avicins were able to overcome this protective effect in co-culture models of MM cells with BM stromal cells and sensitized MM cells to cytotoxic chemotherapy-induced cell death. Using hierarchical clustering analyses and relevance network algorithms, we compared the patterns of MM cell sensitivity to Avicin D vs Avicin G vs. other anti-cancer drugs and found that the pattern of dose-response relationships of the 2 main members of this class of compounds are very similar to each other, but clearly distinct from the patterns of sensitivity or resistance to other drugs, either conventional or investigational. This further supports the notion that the anti-MM properties of Avicins are mediated by molecular mechanisms distinct from those of currently available anti-MM drugs, and also suggests that Avicins may have anti-tumor activity even against subgroups of MM which may be resistant to other novel therapies that are currently in clinical development. These results have provided the framework for ongoing in vivo studies of anti-tumor activity of these agents, to evaluate the feasibility of future clinical trials of Avicins to improve patient outcome in MM.

Polycaprolactone– b-poly(ethylene oxide) copolymer micelles as a delivery vehicle for dihydrotestosterone

Block copolymer micelles formed from copolymers of poly(caprolactone)- b-poly(ethylene oxide) (PCL- b-PEO) were investigated as a drug delivery vehicle for dihydrotestosterone (DHT). The physical parameters of the PCL- b-PEO micelle-incorporated DHT were measured, including the loading capacity of the micelles for DHT, the apparent partition coefficient of DHT between the micelles and the external medium and the kinetics of the release of DHT from the micelle solution. The MTT survival assay was used to assess the in vitro biocompatibility of PCL- b-PEO micelles in HeLa cell cultures. The biological activity of the micelle-incorporated DHT was evaluated in HeLa cells which had been co-transfected with the expression vectors for the androgen receptor and the MMTV-LUC reporter gene. The PCL- b-PEO micelles were found to have a high loading capacity for DHT and the release profile of the drug from the micelle solution was found to be a slow steady release which continued over a 1-month period. The biological activity of the micelle-incorporated DHT was found to be fully retained.

Bax-Dependent Caspase-3 Activation Is a Key Determinant in p53-Induced Apoptosis in Neurons

p53 is a pivotal molecule regulating the death of neurons both after acute injury and during development. The molecular mechanisms by which p53 induces apoptosis in neuronal cells, however, are not well understood. We have shown previously that adenovirus-mediated p53 gene delivery to neurons was sufficient to induce apoptosis. In the present study we have examined the molecular mechanism by which p53 evokes neuronal cell death. Adenovirus-mediated delivery of p53 to cerebellar granule neurons resulted in caspase-3 (CPP32) activation followed by terminal deoxynucleotidyl transferase-mediated biotinylated UTP nick end labeling (TUNEL) staining and loss of viability as determined by an MTT survival assay. To determine whether Bax is essential for caspase-3 activation, p53 was expressed in Bax-deficient cells. Bax null neurons did not exhibit caspase-3 activation in response to p53 and were protected from apoptosis. To determine whether Bax-dependent caspase-3 activation was required in p53-mediated neuronal cell death, caspase-3-deficient neurons were examined. Our results indicate that caspase-3-deficient neurons exhibit a remarkable delay in apoptosis and a dramatic decrease in TUNEL-positive cells. These studies demonstrate that p53-induced cell death in postmitotic neurons involves a Bax-dependent caspase-3 activation, suggesting that these molecules are important determinants in neuronal cell death after injury.