R. S. Rajmani

Canine parvovirus type 2a (CPV-2a)-induced apoptosis in MDCK involves both extrinsic and intrinsic pathways.

The canine parvovirus type 2 (CPV-2) causes an acute disease in dogs. It has been found to induce cell cycle arrest and DNA damage leading to cellular lysis. In this paper, we evaluated the apoptotic potential of the “new CPV-2a” in MDCK cells and elucidated the mechanism of the induction of apoptosis. The exposure of MDCK cells to the virus was found to trigger apoptotic response. Apoptosis was confirmed by phosphatidylserine translocation, DNA fragmentation assays, and cell cycle analysis. Activation of caspases-3, -8, -9, and -12 and decrease in mitochondrial potential in CPV-2a-infected MDCK cells suggested that the CPV-2a-induced apoptosis is caspase dependent involving extrinsic, intrinsic, and endoplasmic reticulum pathways. Increase in p53 and Bax/Bcl2 ratio was also observed in CPV-2a-infected cells.

In-vitro Characterization and Evaluation of Apoptotic Potential of Bicistronic Plasmid Encoding HN Gene of Newcastle Disease Virus and Human TNF-α

The viral gene oncotherapy in combination with cytokines emerges as an exciting strategy for cancer therapy due to its minimal side effects and tumor specificity. HN is the surface protein of NDV which is involved in virus infectivity and is known to kill many cancerous cell types. TNF-α, a multifactorial cytokine has direct anti-tumor activity by activating the extrinsic pathways of apoptosis. In the present study, HN gene of NDV and TNF-α of human were cloned at multiple cloning sites (MCS) 1 and 2 of bicistronic expression vector pVIVO2. Expression pattern of recombinant clone was checked on transcriptional and translational level by RT-PCR, Immunofluorescence assay and flow cytometry. On flow cytometric analysis HN gene expression was found to be 28.30 ± 1.21; 5.22 ± 0.60%, and TNF-α gene expression was found to be 15.44 ± 0.42; 6.51 ± 0.757%, in HeLa cells transfected with pVIVO.nd.hn.hu.tnf and pVIVO2 empty vector control, respectively. These assays confirm that HN and TNF-α act synergistically in the induction of apoptosis in HeLa cells.

Non-Structural protein 1 (NS1) gene of Canine Parvovirus-2 regresses chemically induced skin tumors in Wistar rats

The Non-Structural protein 1 of Canine Parvovirus-2 (CPV2.NS1) plays a major role in viral cytotoxicity and pathogenicity. CPV2.NS1 has been proven to cause apoptosis in HeLa cells in vitro in our laboratory. Here we report that CPV2.NS1 has no toxic side effects on healthy cells but regresses skin tumors in Wistar rats. Histopathological examination of tumor tissue from CPV2.NS1 treated group revealed infiltration of mononuclear and polymorphonuclear cells with increased extra cellular matrix, indicating signs of regression. Tumor regression was also evidenced by significant decrease in mitotic index, AgNOR count and PCNA index, and increase in TUNEL positive apoptotic cells in CPV2.NS1 treated group. Further, CPV2.NS1 induced anti-tumor immune response through significant increase in CD8(+) and NK cell population in CPV2.NS1 treated group. These findings suggest that CPV2.NS1 can be a possible therapeutic candidate as an alternative to chemotherapy for the treatment of cancer.

HN Protein of Newcastle Disease Virus Induces Apoptosis Through SAPK/JNK Pathway.

Many viral proteins are responsible for causing induction of apoptosis in the target cells. Hemagglutinin neuraminidase (HN), a multifunctional protein of Newcastle disease virus (NDV), is one of such proteins. The present study was undertaken to determine the apoptotic potential of the HN gene in cultured human cervical cancer cell line (HeLa cell) and to elucidate the molecular mechanisms involved. The results of the study indicate that HN protein causes apoptosis in HeLa cells, as observed by the translocation of Phosphatidylserine, activation of caspases, cleavage of poly (ADP-ribose) polymerase (PARP), and DNA fragmentation. Further, we report that expression of HN protein upregulates the SAPK/JNK pathway leading to transactivation of c-Jun which in turn activates apoptosis signaling. The results of our study provide an insight into the mechanism through which HN induces apoptosis.

Apoptin as a Potential Viral Gene Oncotherapeutic Agent

The use of viruses for treatment of cancer overcomes the bottlenecks of chemotherapy and radiotherapy. Several viruses and their proteins have been evaluated for oncolytic effect. The VP3 protein (apoptin) of chicken anemia virus is one such protein with an inherent ability to lyse cancer and transformed cells while leaving normal cells unharmed. In the present study, the apoptosis inducing potential of VP3 protein of CAV was evaluated in human cervical cancer cell line (HeLa). It was found that in VP3-induced apoptosis, caspase-dependent intrinsic pathway plays an important role with the cleavage of poly (ADP-ribose) polymerase (PARP) and there was no evidence of involvement of death receptor-mediated extrinsic pathway. The results of this study provide intuitive information and strengthen the candidacy of apoptin as a viral oncotherapeutic agent.

HN protein of Newcastle disease virus sensitizes HeLa cells to TNF-α-induced apoptosis by downregulating NF-κB expression.

Hemagglutinin neuraminidase (HN) is a membrane protein of Newcastle disease virus (NDV) with the ability to induce apoptosis in many transformed cell lines. TNF-α is a multi-factorial protein that regulates cell survival, differentiation and apoptosis. In a previous study, we reported that HN protein induces apoptosis by downregulating NF-κB expression. Further, we speculated that downregulation of NF-κB expression might sensitize HeLa cells to TNF-α-mediated apoptosis. Therefore, the present study was undertaken to investigate if HN protein could sensitize HeLa cells to TNF-α and to examine the apoptotic potential of the HN protein and TNF-α in combination. The results revealed that the pro-apoptotic effects were more pronounced with the combination of HN and TNF-α than with HN or TNF-α alone, which indicates that the HN protein indeed sensitized the HeLa cells to TNF-α-induced cell death. The results of the study provide a mechanistic insight into the apoptotic action of HN protein along with TNF-α, which could be valuable in treating tumor types that are naturally resistant to TNF-α.

Administration of IκB-kinase inhibitor PS1145 enhances apoptosis in DMBA-induced tumor in male Wistar rats.

Nuclear factor kappa‐B (NF‐κB), a key anti‐apoptotic factor, plays a critical role in tumor cell growth, metastasis, and angiogenesis. The transcriptional activity of NF‐κB is normally suppressed in the cytoplasm due to its association with a natural inhibitor molecule IκB. Phosphorylation of the IκB at Ser 32 and Ser 36 by the IκB kinase complex (IKK) marks the degradation of the molecule by 26S proteasome. As NF‐κB is constitutively activated in most of the tumor cells, inhibition of the activities of IKK may significantly sensitize the tumor cells to apoptosis. In the present study, we investigated the effect of IκB kinase‐specific blocker PS1145 on DMBA‐induced skin tumor of male Wistar rats. We examined the apoptotic effect of PS1145 on DMBA‐induced tumor by various histopathological and molecular techniques. Our results demonstrate the significant expression of major pro‐apoptotic genes like caspases 2, 3, 8, 9, and p53 in PS1145‐treated tumor bearing group at mRNA levels as well as significant (P < 0.05) down regulation in the expression levels of NF‐κB and VEGF, the major pro‐inflammatory and pro‐angiogenic factors, respectively. The histopathological examination showed that the tumor progression, mitotic, AgNOR, and PCNA indices were significantly reduced in PS1145 treatment groups as compared to PBS control on day 28 of post‐treatment. Furthermore, significant increase in TUNEL positive nuclei and observation of peculiar apoptotic nuclei in transmission electron microscopy were seen in PS1145 treatment group. We conclude that intravenous application of PS1145 promotes direct apoptosis in DMBA‐induced skin tumor in male Wistar rats by blocking NF‐κB and VEGF activities.

Apoptotic and Immunosuppressive Effects of Turmeric Paste on 7, 12 Di Methyl Benz (a) Anthracene Induced Skin Tumor Model of Wistar Rat

ABSTRACT Dietary components with potent anticancerous property are gaining attention as therapeutic agents due to low cost of therapy and minimal toxic effects. Turmeric is one such miracle spices of Indian and South Asian recipes with multiple medicinal properties. The anticarcinogenic properties of its active compound curcumin have been studied in detail. However, studies on the medicinal properties of crude turmeric used as dietary agents are lacking. Therefore, in this study we investigated the effects of dietary and topical crude turmeric paste on DMBA induced skin tumor of male Wistar rats. We observed the apoptotic effect of crude turmeric paste on DMBA induced tumor with depletion of T cells response. Our results demonstrated the significant expression of major pro-apoptotic genes like caspase-2, 3, 8, 9, PARP, and p53 and down regulation of major pro-inflammatory (NF-κB) and pro-angiogenic factors and (VEGF) in turmeric treated tumor tissues. We also observed significant decrease in CD4+, CD8+, and Natural Killer cell population as compared to the untreated group.