Varsha Kaushal

Autophagy delays apoptosis in renal tubular epithelial cells in cisplatin cytotoxicity

One of the major side effects of cisplatin chemotherapy is toxic acute kidney injury due to preferential accumulation of cisplatin in renal proximal tubule epithelial cells and the subsequent injury to these cells. Apoptosis is known as a major mechanism of cisplatin-induced cell death in renal tubular cells. We have also recently demonstrated that autophagy induction is an immediate response of renal tubular epithelial cell exposure to cisplatin. Inhibition of cisplatin-induced autophagy blocks the formation of autophagosomes and enhances cisplatin-induced caspase-3, -6, and -7 activation, nuclear fragmentation, and apoptosis. The switch from autophagy to apoptosis by autophagic inhibitors suggests that autophagy induction was responsible for a pre-apoptotic lag phase observed on exposure of renal tubular cells to cisplatin. Our studies provide evidence that autophagy induction in response to cisplatin mounts an adaptive response that suppresses and delays apoptosis. The beneficial effect of autophagy has a potential clinical significance in minimizing or preventing cisplatin nephrotoxicity. Addedum to: Yang C, Kaushal V, Shah SV, Kaushal GP. Autophagy and apoptosis are associated in cisplatin injury to renal tubular epithelial cell injury. Am J Physiol Renal Physiol 2008; 294:F777-87.

Transcriptional activation of caspase-6 and -7 genes by cisplatin-induced p53 and its functional significance in cisplatin nephrotoxicity.

This study examined the role of cisplatin-induced p53 activation in regulation of caspases and cellular injury during cisplatin nephrotoxicity. The executioner caspase-6 and -7 but not caspase-3 were identified as transcriptional targets of p53 in cisplatin injury as revealed by chromatin immunoprecipitation, a reporter gene and electrophoretic mobility shift assays, and real-time PCR following overexpression and inhibition of p53. DNA binding by p53 involved the first introns of the human and mouse caspase-7 gene and the mouse caspase-6 gene. Studies in human kidney, breast, ovary, colon, and prostate tumor cell lines also validated these findings. Treatment of p53 (−/−) cells with cisplatin did not induce caspase-6 and -7 expression and subsequent activation. In caspase-3 (−/−) cells, inhibition of caspase-6 and -7 activations markedly prevented cisplatin-induced cell death. In an in vivo model of cisplatin nephrotoxicity inhibition of p53 activation by a p53 inhibitor suppressed transactivation of the caspase-6 and -7 genes and prevented renal failure. p53 (−/−) mice were resistant to cisplatin nephrotoxicity as assessed by renal function and histology. These studies provide first evidence for p53-dependent transcriptional control of the caspase-6 and -7 genes and its functional significance in cisplatin injury to renal cells and functional implication of cisplatin-induced p53 induction in vitro and in vivo in cisplatin nephrotoxicity.

Thalidomide protects endothelial cells from doxorubicin‐induced apoptosis but alters cell morphology

Summary.  Antiangiogenesis agents are now being used in clinical trials to reduce the risk of recurrence of cancer. Several of these agents, however, are associated with thrombosis, especially when used in combination with chemotherapy. Antiangiogenesis and thrombosis are both endothelial‐related activities, and we therefore evaluated one presumed antiangiogenesis agent (thalidomide) on intact cultured endothelial cells, and on cultured endothelial cells injured by preincubation with doxorubicin. We evaluated cell viability, caspase‐3 activation, morphology of cells using light microscopy, and protease activated receptor‐1 (PAR‐l) expression. In our experiments, doxorubicin induced a dose‐ and incubation time‐dependent and caspase‐3‐mediated apoptosis of endothelial cells. Thalidomide alone caused no changes in intact endothelial cells in terms of morphology, cell viability or activation of caspase‐3. In contrast, when thalidomide was added to doxorubicin‐injured endothelial cells, there was protection from cell death, increase in viability of endothelial cells, induction of differentiation and formation of neotubules. Doxorubicin reduced the expression of thrombin receptor, PAR‐1, as evaluated by immunostaining and flow cytometry. Thalidomide did not alter PAR‐1 expression in untreated cells but restored its expression reduced by doxorubicin. These findings suggest that thalidomide may be procoagulant, not by enhancing doxorubicin‐mediated endothelial cell injury, but by altering the expression of PAR‐1 on injured endothelium and resulting in endothelial dysfunction, which may explain hypercoagulability in patients treated with chemotherapy followed by thalidomide.

Meprin A and meprin α generate biologically functional IL-1β from pro-IL-1β

The present study demonstrates that both oligomeric metalloendopeptidase meprin A purified from kidney cortex and recombinant meprin alpha are capable of generating biologically active IL-1beta from its precursor pro-IL-1beta. Amino-acid sequencing analysis reveals that meprin A and meprin alpha cleave pro-IL-1beta at the His(115)-Asp(116) bond, which is one amino acid N-terminal to the caspase-1 cleavage site and five amino acids C-terminal to the meprin beta site. The biological activity of the pro-IL-1beta cleaved product produced by meprin A, determined by proliferative response of helper T-cells, was 3-fold higher to that of the IL-1beta product produced by meprin beta or caspase-1. In a mouse model of sepsis induced by cecal ligation puncture that results in elevated levels of serum IL-1beta, meprin inhibitor actinonin significantly reduces levels of serum IL-1beta. Meprin A and meprin alpha may therefore play a critical role in the production of active IL-1beta during inflammation and tissue injury.

Expression of tissue factor in prostate cancer correlates with malignant phenotype

Tissue factor (TF), apart from its established role in hemostasis, has been implicated in promoting angiogenesis and metastasis in a wide array of tumors including prostate cancer. Expression of TF was evaluated in freshly-resected prostate specimens obtained from patients with localized (n=9) and androgen ablated (n=6) disease using real-time reverse transcription-polymerase chain reaction and Western blot analysis. TF was detected in all specimens in both stages of the disease. We further analyzed for correlations between TF expression and those of several angiogenic growth factors and their receptors. TF RNA expression correlated significantly with expression of vascular endothelial growth factor-A in these specimens (s=0.621, P=0.013). Eighty-one prostate specimens from patients with benign prostatic hyperplasia (n=27), localized prostate cancer (ES, n=32), and advanced disease (n=22) were also evaluated using immunohistochemistry and findings were correlated with clinical parameters. TF expression was detected on epithelial cells of the malignant glands. Furthermore, its expression levels correlated significantly with Gleason score (s=0.58, P=0.0001) and with the stage of the disease (s=0.441, P=0.0001) in these specimens. These data support the role of TF in angiogenesis and disease progression.

Potential anticancer effects of statins: fact or fiction?

Deregulation of any of the steps in cell growth, proliferation and apoptosis may result in its malignant transformation. Statins, along with their lipid-lowering potential, modify several processes in the cell cycle. These agents inhibit cell proliferation and arrest cell cycle progression by interrupting growth-promoting signals. Statins selectively induce proapoptotic protential in tumor cells and synergistically enhance proapoptotic potential of several cytotoxic agents. Statins alter angiogenic potential of cells by modulating apoptosis inhibitory effects of VEGF and decrease secretion of metalloproteases. Statins also alter adhesion and migration of tumor cells, thereby inhibiting tumor invasion and metastasis. Statins suppress rate of activation of multiple coagulation factors and thus prevent coagulation-mediated angiogenesis. Statins have been shown to have anti-tumor activity in experimental models. Various anti-neoplastic properties of statins are probably a result of inhibition of posttranslational modifications of growth regulatory proteins. Molecular mechanisms of antiproliferative, proapoptotic and antiangiogenic effects of statins are reviewed in this chapter.

Prospective study of circulating angiogenic markers in prostate-specific antigen (PSA)-stable and PSA-progressive hormone-sensitive advanced prostate cancer.

OBJECTIVES To prospectively describe and compare circulating vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) in two groups of advanced prostate cancer patients undergoing androgen deprivation. The first patient group (n = 21) consisted of patients with stable serum prostate-specific antigen (PSA) and the second group (n = 20) consisted of patients with a rising serum PSA during androgen deprivation. METHODS Patients with diabetes or active heart disease or those receiving anticoagulants were excluded. Circulating VEGF and bFGF were measured in platelet-poor plasma. bFGF was also measured in urine. Platelet factor 4 protein (PF4) assays were performed to evaluate platelet activity in platelet-poor plasma samples. Commercially available enzyme-linked immunosorbent assay kits were used for all assays, and all tests were performed in duplicate. RESULTS The median age of this study population was 75 years (range 58 to 85). Median plasma VEGF measured in the PSA-stable group was 801.5 pg/mL and in the PSA-rising group was 655.5 pg/mL (P = 0.464). Circulating bFGF was undetectable in plasma, but 4 patients in the PSA-stable group had measurable urine levels. Platelet-poor plasma PF4 assays in all patients were less than 3 IU/mL (normal range 0 to 10). CONCLUSIONS Our pilot study suggests elevated plasma VEGF levels in advanced prostate cancer do not increase during failure of androgen deprivation therapy. Most of the advanced cancer patients in this study expressed plasma VEGF. This suggests its potential role as a surrogate marker for response assessment during antiangiogenic therapy in this stage.

Differential Toxicity of Anthracyclines on Cultured Endothelial Cells

Anthracyclines are known for their endothelial toxicity. Newer derivatives may have fewer toxic effects on endothelium. The authors therefore evaluated the effects of doxorubicin, doxorubicin analogs (daunorubicin, idarubicin), and pegylated liposomal doxorubicin (doxil) in human coronary artery endothelial cells (HCAECs). Endothelial viability did not change significantly with doxil, but was decreased with doxorubicin, daunorubicin, or idamycin. Similarly caspase-3 activity was significantly elevated in HCAECs treated with doxorubicin, daunorubicin, and idamycin. In contrast, doxil did not cause significant increase in caspase activity. The authors also characterized the levels of antiapoptotic and prosurvival proteins using Western blot analysis. There was no significant difference in the expression levels of Bcl-2, Bax, and phospho-Akt in endothelial cells treated with anthracycline derivatives. However, the expression levels of Mcl-l protein were unaltered in endothelial cells treated with doxil but were significantly decreased when treated with other anthracycline analogs. Doxil minimally affected the expression levels of p53, whereas other anthracyclines induced p53 protein levels to a significant level, resulting in endothelial cell apoptosis. The authors conclude that the liposomal anthracycline protects endothelial cells from injury by preventing caspase-3 activation and maintaining the expression of antiapoptotic molecule Mcl-1.

Caspase Protocols in Mice

Members of the caspase family of proteases are evolutionarily conserved cysteine proteases that play a crucial role as the central executioners of the apoptotic pathway. Since the discovery of caspases, many methods have been developed to detect their activation and are widely used in basic and clinical studies. In a mouse tissue, caspase activation can be monitored by cleavage of caspase-specific synthetic substrates and by detecting cleaved caspase by western blot analysis of the tissue extract. In tissue sections, active caspase can be detected by immunostaining using specific antibodies to the active caspase. In addition, among the myriads of caspase-specific substrates known so far, cleaved fragments produced by caspases from the substrates such as PARP, lamin A, and cytokeratin-18 can be monitored in tissue sections by immunostaining as well as western blots of tissue extracts. In general, more than one method should be used to ascertain detection of activation of caspases in a mouse tissue.

Cyclooxygenase-2 Expression in Prostate Cancer: An Inconsistent Therapeutic Target

Objectives: To determine cyclooxygenase-2 (COX-2) expression patterns in primary prostate tissue of patients undergoing androgen deprivation therapy and in patients with early stage disease. Methods: Gene and protein expression analysis were performed in freshly resected prostate cancer tissue obtained from patients with advanced stage cancer who presented with bladder obstruction Local stage cancer specimens were collected from patients undergoing radical prostatectomy (RP) Expression studies in fresh specimens included quantitative real time polymerase chain reaction, western immunoblotting and immunohistochemistry (IHC). Mean COX-2 gene expressions were compared in both stages. In addition to evaluating protein expression by IHC in fresh specimens, an unrelated archival specimen set of 23 additional clinically matched, advanced and 20 local stage cancer specimens were also analyzed. The pooled (fresh and archival) specimen set ( each) was then used for evaluating protein expression by IHC. Thirty non-c...