Biomarkers and Redox Balance in Aging Rats after Dynamic and Isometric Resistance Training.

Aging muscle is prone to sarcopenia and its associated telomere shortening and increased oxidative stress. Telomeres are protected by a shelterin protein complex, proteins expressed in response to DNA damage. Aerobic exercise training has shown to positively modulate these proteins while aging, but the effects of resistance training are less clear. This investigation was to examine the role of dynamic and isometric RT on markers of senescence and muscle apoptosis: checkpoint kinase 2, 53 kDa protein, shelterin telomere repeat binding 1 and 2, DNA repair, telomere length and redox state in the quadriceps muscle. Fifteen 49-week-old male rats were divided into three groups: control, dynamic resistance training, and isometric resistance training. Dynamic and isometric groups completed five sessions per week during 16 weeks at low to moderate intensity (20-70% maximal load). Only dynamic group decreased expression of 53 kDa protein, proteins from shelterin complex, oxidative stress, and improved antioxidant defense. There was no difference among groups regarding telomere length. In conclusion, dynamic resistance training was more effective than isometric in reducing markers of aging and muscle apoptosis in elderly rats. This modality should be considered as valuable tool do counteract the deleterious effects of aging.

Immunohistochemistry analysis of checkpoint kinase 2 in oral squamous cell carcinoma

Background: Oral squamous cell carcinoma (OSCC) is the most frequently occurring malignant tumor of the head and neck region. Chk2 (Checkpoint kinase 2) is considered a tumor suppressor gene that acts on the cellular response to DNA damage. However, the role of Chk2 in OSCC prognosis is not yet fully understood. The objective of this study was to evaluate Chk2 immunoexpression in OSCC and to elucidate the association between its expression and clinicopathological parameters of prognostic importance, including overall survival, disease-free survival, and metastasis-free survival. Methods: Chk2 expression was analyzed in 101 samples from patients with OSCC using immunohistochemistry. We stratified the patients into high expression (> 66% of cells positive for Chk2) and low expression (< 66%) groups. Results: Chk2 showed high expression in 57.43% of OSCC. In our study, the expression of Chk2 did not correlate with any of the prognostic parameters evaluated. There was no difference between overall survival, metastasis-free survival, and disease-free survival according to Chk2 expression. Conclusion: Despite the great importance of Chk2 in the development of different types of cancer, our findings do not favor Chk2 as a prognostic marker in oral squamous cell carcinoma.

Induction of p53 Phosphorylation at Serine 20 by Resveratrol Is Required to Activate p53 Target Genes, Restoring Apoptosis in MCF-7 Cells Resistant to Cisplatin.

Resistance to cisplatin (CDDP) is a major cause of cancer treatment failure, including human breast cancer. The tumor suppressor protein p53 is a key factor in the induction of cell cycle arrest, DNA repair, and apoptosis in response to cellular stimuli. This protein is phosphorylated in serine 15 and serine 20 during DNA damage repair or in serine 46 to induce apoptosis. Resveratrol (Resv) is a natural compound representing a promising chemosensitizer for cancer treatment that has been shown to sensitize tumor cells through upregulation and phosphorylation of p53 and inhibition of RAD51. We developed a CDDP-resistant MCF-7 cell line variant (MCF-7R) to investigate the effect of Resv in vitro in combination with CDDP over the role of p53 in overcoming CDDP resistance in MCF-7R cells. We have shown that Resv induces sensitivity to CDDP in MCF-7 and MCF-7R cells and that the downregulation of p53 protein expression and inhibition of p53 protein activity enhances resistance to CDDP in both cell lines. On the other hand, we found that Resv induces serine 20 (S20) phosphorylation in chemoresistant cells to activate p53 target genes such as PUMA and BAX, restoring apoptosis. It also changed the ratio between BCL-2 and BAX, where BCL-2 protein expression was decreased and at the same time BAX protein was increased. Interestingly, Resv attenuates CDDP-induced p53 phosphorylation in serine 15 (S15) and serine 46 (S46) probably through dephosphorylation and deactivation of ATM. It also activates different kinases, such as CK1, CHK2, and AMPK to induce phosphorylation of p53 in S20, suggesting a novel mechanism of p53 activation and chemosensitization to CDDP.

Modulation of RhoA GTPase Activity Sensitizes Human Cervix Carcinoma Cells to γ-Radiation by Attenuating DNA Repair Pathways.

Radiotherapy with γ-radiation is widely used in cancer treatment to induce DNA damage reducing cell proliferation and to kill tumor cells. Although RhoA GTPase overexpression/hyperactivation is observed in many malignancies, the effect of RhoA activity modulation on cancer radiosensitivity has not been previously investigated. Here, we generated stable HeLa cell clones expressing either the dominant negative RhoA-N19 or the constitutively active RhoA-V14 and compared the responses of these cell lines with those of parental HeLa cells, after treatment with low doses of γ-radiation. HeLa-RhoA-N19 and HeLa-RhoA-V14 clones displayed reduced proliferation and survival compared to parental cells after radiation and became arrested at cell cycle stages correlated with increased cellular senescence and apoptosis. Also, Chk1/Chk2 and histone H2A phosphorylation data, as well as comet assays, suggest that the levels of DNA damage and DNA repair activation and efficiency in HeLa cell lines are correlated with active RhoA. In agreement with these results, RhoA inhibition by C3 toxin expression drastically affected homologous recombination (HR) and nonhomologous end joining (NHEJ). These data suggest that modulation of RhoA GTPase activity impairs DNA damage repair, increasing HeLa cell radiosensitivity.

Resveratrol abrogates the temozolomide-induced G2 arrest leading to mitotic catastrophe and reinforces the temozolomide-induced senescence in glioma cells.

BACKGROUND: Temozolomide (TMZ) is the most widely used drug to treat glioblastoma (GBM), which is the most common and aggressive primary tumor of the Central Nervous System and one of the hardest challenges in oncotherapy. TMZ is an alkylating agent that induces autophagy, apoptosis and senescence in GBM cells. However, therapy with TMZ increases survival after diagnosis only from 12 to 14.4 months, making the development of combined therapies to treat GBM fundamental. One candidate for GBM therapy is Resveratrol (Rsv), which has additive toxicity with TMZ in several glioma cells in vitro and in vivo. However, the mechanism of Rsv and TMZ additive toxicity, which is the aim of the present work, is not clear, especially concerning cell cycle dynamics and long term effects. METHODS: Glioma cell lines were treated with Rsv and TMZ, alone or in combinations, and the induction and the role of autophagy, apoptosis, cell cycle dynamics, protein expression and phosphorylation status were measured. We further evaluated the long term senescence induction and clonogenic capacity. RESULTS: As expected, temozolomide caused a G2 cell cycle arrest and extensive DNA damage response. Rsv did not reduced this response, even increasing pATM, pChk2 and gammaH2Ax levels, but abrogated the temozolomide-induced G2 arrest, increasing levels of cyclin B and pRb(S807/811) and reducing levels of pWee1(S642) and pCdk1(Y15). This suggests a cellular state of forced passage through G2 checkpoint despite large DNA damage, a scenario that may produce mitotic catastrophe. Indeed, the proportion of cells with high nuclear irregularity increased from 6 to 26% in 48 h after cotreatment. At a long term, a reduction in clonogenic capacity was observed, accompanied by a large induction of senescence. CONCLUSION: The presence of Rsv forces cells treated with TMZ through mitosis leading to mitotic catastrophe and senescence, reducing the clonogenic capacity of glioma cells and increasing the chronic effects of temozolomide.