ABSTRACT
The combined antiretroviral therapy (cART), a multidrug combination regimen, usually consisting Nucleoside Reverse Transcriptase Inhibitors, non- Nucleoside Reverse Transcriptase Inhibitors and Protease Inhibitors has altered the morbidity pattern affecting HIV-infected individuals to include non-AIDS-defining malignancies (nADMs). The speculation is rife; does cART induce or promote the progression of nADMs such as breast cancer? This study was therefore designed to investigate of the effects of some antiretroviral drugs (at clinically relevant concentrations) on the expression of anti-angiogenic gene; VEGF165b in two human breast cell lines; MCF-7 and MCF-10A by Real Time qPCR and immuno-fluorescence. All of the antiretroviral drugs and combinations tested produced patterns of slight up or downregulation of VEGF165b mRNA expression but the alterations did not attain statistical significance. They also did not alter VEGF165bprotein localisation in both cell lines. The findings reported here suggest that antiretroviral drugs probably do not influence the angiogenic pathway in the development of breast cancer in patients under the combined antiretroviral regimen.
El tratamiento antirretroviral combinado (TARc), un régimen de combinación de múltiples fármacos, consistiendo generalmente en inhibidores nucleósidos de la transcriptasa reversa, inhibidores no-nucleósidos de la transcriptasa reversa e inhibodres de proteasa que alteran el patrón de mortalidad que afecta a infectados por el VIH incluyendo neoplasias definidas como no HIV (nADMs). La especulación es moneda corriente; TARc induce o promueve la progresión de nADMs como cáncer de mama? Por lo tanto, este estudio se diseñó para investigar los efectos de algunos de los fármacos antirretrovirales (en concentraciones clínicamente relevantes) sobre la expresión del gen anti-angiogénico; VEGF165b en dos líneas celulares de mama humana; MCF-7 y MCF-10A por PCR tiempo real e inmunofluorescencia. Todos los fármacos antirretrovirales y las combinaciones probadas pueden regular en forma ligera hacia arriba o hacia abajo la expresión de ARNm producidos por VEGF165b pero las alteraciones no fueron estadísticamente significativos. Además, no se alteran los niveles de proteína VEGF165b, para la localización en ambas líneas celulares. Los resultados aquí presentados sugieren que los medicamentos antirretrovirales probablemente no influyen en la vía angiogénica en el desarrollo del cáncer de mama en pacientes bajo el régimen antirretroviral combinado.
Subject(s)
Humans , Female , Adenocarcinoma/metabolism , Angiogenesis Inhibitors/pharmacology , Breast Neoplasms/metabolism , Protease Inhibitors/pharmacology , Reverse Transcriptase Inhibitors/pharmacology , Epithelial Cells , Immunohistochemistry , MCF-7 Cells , Polymerase Chain Reaction , Vascular Endothelial Growth Factor AABSTRACT
Apoptosis is a tightly programmed cell suicide which occurs in multiple physiologic and pathological conditions where it plays an important role in tissue development and homeostasis by eliminating unwanted and damaged cells. Appropriate apoptosis signalling is crucial in maintaining the fine balance between cell death and cell survival in cancer. In response to death stimuli the morphology of the cell undergoes unique changes. The aim of this study was to examine and compare the changes in the cell surface morphology using scanning electron microscopy in HCS-2 cells, following 24 hour treatment with components of highly active antiretroviral therapy (HAART) at their clinical plasma concentrations. The cells were fixed in 2.5% Glutaraldehyde and post-fixed in 1% osmium tetroxide. The cells were then dehydrated through a graded series of alcohol and treated with hexamethyl-disilazane, then coated with a double layer of carbon. The cells were viewed under a Zeiss Ultra FEG Scanning Electron Microscope and a one way ANOVA and Tukey Kramer Post Hoc test was conducted based on the scoring of surface morphology of the cells using JMP 11 statistical software. The drugs used in this study induced morphological features which are known to be characteristic of apoptotic cell death. The drug combinations (ATP and LPV/r) were seemingly more effective than individual treatments in inducing cell death because morphological features observed were more advanced than those observed in individual treatments. However, LPV/r was more potent than ATP. In conclusion, HAART showed anticancer properties by inducing cell death through apoptosis.
ABSTRACT
BACKGROUND: Methanol and DMSO are commonly used as carrier solvents for lipophilic chemicals in in-vitro experiments. However, very little information is available regarding the effects of these solvents on the expression of pro and anti-apoptotic genes and proteins. MATERIALS AND METHODS: In this study, we examined the cytotoxic effects of methanol and dimethylsulfoxide at 0.5% (final concentrations recommended for in-vitro toxicity assays) on human breast cancer MCF-7 cells. We also investigated the effects of these solvents on the mRNA and immunocytochemical expression of apoptotic proteins BAX and BCL-2. RESULTS: The results of neutral red cell viability assay showed that methanol and DMSO concentrations of 0.5% exhibited no cytotoxic effects on MCF-7 cells following a 24 hour exposure. Gene expression and Immunofluorescence results showed that methanol but not DMSO reduced the expression of the BAX pro-apoptotic protein, while both solvents did not alter the expression of the BCL-2 oncoprotein. CONCLUSION: Our results suggest that while methanol concentrations at 0.5% may be appropriate for in vitro toxicity studies in human breast cancer MCF-7 cells, it could alter the results of gene and protein expression experiments.
ABSTRACT
Cervical cancer is the third most commonly diagnosed cancer globally and it is one of three AIDS defining malignancies. Highly active antiretroviral therapy (HAART) is a combination of three or more antiretroviral drugs and has been shown to play a significant role in reducing the incidence of some AIDS defining malignancies, although its effect on cervical cancer is still unclear. The aim of this study was to investigate the relationship between cervical cancer and HAART. This was achieved by studying the expression of two signalling molecules expressed in cervical cancer; MUC1 and P65. Following the 24-hour treatment of a cervical cancer cell line, HCS-2, with drugs, which are commonly used as part of HAART at their clinical plasma concentrations, real-time qPCR and immunofluorescence were used in order to study gene and protein expression. A one-way ANOVA followed by a Tukey-Kramer post-hoc test was conducted using JMP 11 software on both sets of data. The drug classified as a protease inhibitor (PI) (i.e. LPV/r) reduced MUC1 and P65 gene and protein expression more than the other drug tested. PIs are known to play a significant role in cell death; therefore, the cells were thought to be more susceptible to cell death following treatment with PIs. In conclusion, the drugs used, especially the PI showed some anticancer effects by facilitating cell death through decreased gene and protein expression of MUC1 and P65 and present promising agents for cancer treatment.
Subject(s)
Antiretroviral Therapy, Highly Active , Mucin-1/genetics , Transcription Factor RelA/genetics , Uterine Cervical Neoplasms/drug therapy , Uterine Cervical Neoplasms/genetics , Anti-HIV Agents/blood , Anti-HIV Agents/therapeutic use , Cell Line, Tumor , Female , Fluorescent Antibody Technique , Gene Expression Regulation, Neoplastic , Humans , Mucin-1/metabolism , Transcription Factor RelA/metabolism , Uterine Cervical Neoplasms/bloodABSTRACT
Protease inhibitors (PIs) and reverse transcriptase drugs are important components of highly active antiretroviral therapy (HAART) for treating human acquired immunodeficiency syndrome (AIDS). Long-term clinical therapeutic efficacy and treatment compliance of these agents have been limited by undesirable adverse effects and their oncogenicity has been queried. This study investigated the effects of selected antiretroviral agents on the expression of key apoptotic regulatory genes; Bax and Bcl-2 in two cervical cell lines HCS-2 and NCE16IIA by real-time qPCR gene expression and immunocytochemistry. The anti-apoptotic effects of the PI-LPV/r were investigated by cell death detection ELISA and acridine orange staining. All the antiretroviral drugs and combinations tested had no effects on Bax and Bcl-2 gene expression and protein localisation in both cell lines. The protease inhibitors-LPV/r exhibited significant (P<0.05) inhibition of camptothecin-induced apoptosis in the cervical cancer HCS-2 cell line but not in the normal immortalised NCE16IIA cell line. This anti-apoptotic property of HIV protease inhibitors, although shown so far not to involve protein and RNA synthesis might promote the development of cancer.
