nitrophenols and Osteosarcoma

ABT-737 is a BH-3 mimetic that inhibits Bcl-2 and induces apoptosis of cancer cells, which has potential for anticancer therapies. Studies have shown that Bcl-2 expression in human osteosarcoma (OS) cells plays a significant role in tumor progression; however, its effects on OS cell apoptosis are still unknown. Therefore, we examined whether ABT-737 was effective in eliminating human U-2OS cells, either alone or in combination with the chemotherapy drug cisplatin [cis-diamminedichloroplatinum (II); DDP]. Furthermore, we studied the molecular mechanisms of ABT-737 in combination with DDP to induce apoptosis. To analyze the role of ABT-737 and/or DDP on osteosarcoma progression, CCK-8 viability assay, flow cytometry, Hoechst 33258 staining, and western blots were performed. Combined use of ABT-737 and DDP synergistically suppressed cell viability and induced apoptosis in human U-2OS cells when compared with either compound treated alone at low doses. We found that the combination of ABT-737 and DDP upregulated the expression of the pro-apoptotic protein Bax and downregulated the expression of the pro-survival protein Bcl-2, resulting in a change in the Bax/Bcl-2 ratio, release of cytochrome c, and activation of the mitochondrial apoptotic pathway, which resulted in caspase-9 and caspase-3 activation and PARP cleavage. Our results demonstrated that ABT-737 alone has a nominal influence on human U-2OS cells when treated within the clinically administered range, but when combined with DDP, it can inhibit the proliferation of human U-2OS cells by inducing apoptosis via the mitochondrial apoptotic pathway.

Topics: Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Biphenyl Compounds; Cell Line, Tumor; Cell Proliferation; Cell Survival; Cisplatin; Gene Expression Regulation, Neoplastic; Humans; Mitochondria; Neoplasm Proteins; Nitrophenols; Osteosarcoma; Piperazines; Signal Transduction; Sulfonamides

High-grade conventional osteosarcoma is the most common primary bone tumor. Prognosis for osteosarcoma patients is poor and resistance to chemotherapy is common. We performed an siRNA screen targeting members of the Bcl-2 family in human osteosarcoma cell lines to identify critical regulators of osteosarcoma cell survival. Silencing the anti-apoptotic family member Bcl-xL but also the pro-apoptotic member Bak using a SMARTpool of siRNAs as well as 4/4 individual siRNAs caused loss of viability. Loss of Bak impaired cell cycle progression and triggered autophagy. Instead, silencing Bcl-xL induced apoptotic cell death. Bcl-xL was expressed in clinical osteosarcoma samples but mRNA or protein levels did not significantly correlate with therapy response or survival. Nevertheless, pharmacological inhibition of a range of Bcl-2 family members showed that inhibitors targeting Bcl-xL synergistically enhanced the response to the chemotherapeutic agent, doxorubicin. Indeed, in osteosarcoma cells strongly expressing Bcl-xL, the Bcl-xL-selective BH3 mimetic, WEHI-539 potently enhanced apoptosis in the presence of low doses of doxorubicin. Our results identify Bcl-xL as a candidate drug target for sensitization to chemotherapy in patients with osteosarcoma.

Topics: Antineoplastic Combined Chemotherapy Protocols; Apoptosis; bcl-X Protein; Benzopyrans; Biphenyl Compounds; Bone Neoplasms; Cell Line, Tumor; Cohort Studies; Doxorubicin; Drug Synergism; Enzyme Inhibitors; Humans; Immunohistochemistry; Lung Neoplasms; Nitriles; Nitrophenols; Osteosarcoma; Piperazines; Sulfonamides; Tissue Array Analysis; Transfection

Alkaline phosphatase (ALP) is the most widely recognized biochemical marker for osteoblast activity. Although its precise function is poorly understood, it is believed to play a role in skeletal mineralization. The aim of this study was to develop an assay suitable for measuring the activity of this enzyme in microtiter plate format. Using the well-characterized osteoblast-like cell line Saos-2, this paper describes an optimized biochemical assay suitable for measuring ALP activity in tissue culture samples. We have determined that a p-nitrophenyl phosphate substrate concentration of 9 mM provides highest enzyme activities. We have found that cell concentration, and hence enzyme concentration, affects both the kinetics and precision of the assay. We also tested several methods of enzyme solubilization and found that freeze-thawing the membrane fractions twice at -70 degrees C/37 degrees C or freeze-thawing once with sonication yielded highest enzyme activities. The activity of the enzyme decreased by 10% after 7 days storage. This assay provides a sensitive and reproducible method that is ideally suited for measuring ALP activity in isolated osteoblastic cells, although sample preparation and storage can influence results.

Topics: Alkaline Phosphatase; Analysis of Variance; Bone Neoplasms; Buffers; Cell Count; Humans; Indicators and Reagents; Kinetics; Nitrophenols; Organophosphorus Compounds; Osteoblasts; Osteosarcoma; Reference Standards; Reproducibility of Results; Substrate Specificity; Tumor Cells, Cultured