MDA7 is a Tumor Selective Apoptosis Inducing Factor

Chemo Secrets From a Breast Cancer Survivor

Breast Cancer Survivors

Get Instant Access

The initial observation of mda-7 loss of expression in melanomas and its correlation with progression of this type of tumors suggested growth suppressive properties in melanoma cells (4-7). Ensuing studies investigated the effects of ectopic expression of mda-7 in a wide variety of tumor cells (melanoma, carcinomas of the breast, colon, prostate, nasopharynx, high grade gliomas, and osteosarcoma) and proved that mda-7 inhibits tumor cell growth regardless of the status of other genes (p53, Rb, Bax or p16) (1,9,10) (see Fig. 2 for a representative study of lung cancer cells). However, expression of the gene in normal human skin fibroblasts and mammary breast epithelial cells did not significantly affect their growth or trigger apoptosis (9,10). Together, these reports indicate that MDA-7 is an IL-10 family cytokine with tumor cell apoptotic activity and that the cytotoxic effects it induces are specific to tumor cells (5,10,22-24). Several studies have investigated the signal transduction pathways that mediate the apoptotic activity of mda-7. These appear to be multiple, cell-type specific, and include effects induced by the intracellular form of the protein, and by the secreted form (bystander effect) (14).

Although activation of several apoptotic mediators (BAX, BAK, TRAIL, p53, Fas, and DR4), and signaling molecules (PKR, p38MAPK, PI3K, JNK, GSK-3) have been reported, all these signaling molecules appear to converge on the common death effector mechanism mediated by caspase activation and mitochondrial destruction. In lung tumor cells, Ad-mda7-induced apoptosis is mediated by the release of cytochrome c (cyt c) and activation of a caspase 9/Apaf1/cyt c complex (apoptosome), which may involve cell death receptors (14,19,28). Inhibition of caspase activation using ZVAD partially blocked apoptosis (see Fig. 3). Cytosolic cyt c is an essential part of the apop-tosome, which activates caspase 9, which in turn activates other caspases (including caspase 3), triggering apoptosis. In lung cancer cells transduced with Ad-mda7, sharp increases in cytosolic cyt c levels were followed by induction of apoptosis, and occurred without changes in the mitochondrial membrane potential (28). Staurosporine treatment

Fig. 2. Ad-mda7 kills lung tumor cells. Four NSCLC cell lines (H1299, H460, H322, and H358) were treated with Ad-luc (black bars) or Ad-mda7 (gray bars) at 2000 vp/cell and cell proliferation assayed at days 3 and 5 using tritiated thymidine assay. Data are shown as mean+SD.

was used as a positive control to induce cyt c release via mitochondrial permeability transition-dependent pores (MPTPs), and cyclosporine used to specifically inhibit MPTP. The authors report that Ad-mda7-induced cyt c release from the mitochondria was not blocked by cyclosporine, unlike that caused by treatment with staurosporine or Ad-p53. Western analysis of lysates from MDA-7 expressing cells showed no alterations in the levels of BAX, BAK, or Bcl-2, TNF-a, TNF-R1, or TNF-receptor associated death domain protein (TRADD), which are known to act via MPTPs; but pointed to a significant increase in FasL, which can activate caspase 8 and induce cleavage of Bid. This suggested that an MPTP-independent pathway is involved in the apoptotic effects induced by Ad-mda7. The effect, the authors hypothesized, could be mediated by an extrinsic death receptor pathway, involving activation of the IFN-inducible double-stranded RNA protein kinase (PKR), and other protein intermediaries. This hypothesis was in agreement with the results of a separate report by Pataer et al. which puts forward evidence for the key role of PKR in the apoptotic effects induced by MDA-7 expression (29). In this study, infection with Ad-mda7 did not activate apoptosis when PKR function was inhibited by treatment with 2-AP (a specific serine and threonine kinase inhibitor). The need of a functional PKR pathway was further corroborated by Ad-mda7 transduction of PKR-null and PKR wild-type MEFs: apoptosis was absent in the PKR-null cells, but observed at high levels in their wild type PKR counterparts (29).

In breast cancer cells, infection with Ad-mda7 results in a clear and dramatic increase of the proapoptotic protein BAX, as compared with normal human mammary epithelial cells (HMEC), and the ratio of BAX to antiapoptotic protein Bcl-2 becomes significantly higher (9,24). The up-regulation of a proapoptotic member of the Bcl-2 family is of particular interest in breast cancer cells because estrogens regulate Bcl-2 gene expression in mammary epithelial cells and estrogen receptor (ER)-positive breast cancer lines (24,30,31). Bcl-2 family members share four conserved motifs, Bcl-2

Fig. 3. Ad-mda7 induces apoptosis. H1299 NSCLC and T47D breast cancer cells were treated with Ad-luc or Ad-mda7 (2000 vp/cell) either alone or in presence of ZVAD (0.1 ug/mL). Apoptosis induction was assessed at day 3 using Annexin V assay. ZVAD significantly reduces apoptosis, but does not completely block it. Data are shown as mean+SD.

Fig. 3. Ad-mda7 induces apoptosis. H1299 NSCLC and T47D breast cancer cells were treated with Ad-luc or Ad-mda7 (2000 vp/cell) either alone or in presence of ZVAD (0.1 ug/mL). Apoptosis induction was assessed at day 3 using Annexin V assay. ZVAD significantly reduces apoptosis, but does not completely block it. Data are shown as mean+SD.

homology domains (BH-1 to BH-4); whereas antiapoptotic family members contain all four, proapoptotic members have only BH-3. The BH-3 domain is essential to one of their death-regulatory functions given that Bcl-2 proteins are known to homo- and hetero-dimerize with each other, and this domain acts as a peptide ligand that allows for the interaction (32,33). Other death-related functions of this family of proteins are dimerization-independent: binding of the CED-4-like domain of Apaf-1, which prevents its association with pro-caspase 9, and its activation; and pore-forming capabilities that could induce alterations in mitochondrial permeability and thus the release of caspase-activating proteins like cyt c (34-37). Bcl-2 family proteins act at the crossroads of several upstream pro- and antiapoptotic pathways. Thus, reports of mda-7-induced alterations in the level or activity of these proteins are in agreement with preliminary studies, and also with published studies by our group, showing that Ad-mda7 transduction significantly enhances the response of breast cancer cells to radiation therapy, and to a variety of chemotherapeutic agents (tamoxifen, docetaxel, adri-amycin, and herceptin), in spite of their diverse mechanisms of action. Furthermore, the cancer growth inhibitory effects of Ad-mda7 expression were corroborated in vivo (38,39).

Was this article helpful?

0 0
Smoking Solutions

Smoking Solutions

How To Maintain The Stop Smoking Pledge From Your New Year’s Resolution. Get All The Support And Guidance You Need To Be A Success At Quitting Smoking. This Book Is One Of The Most Valuable Resources In The World When It Comes To How To Maintain The Stop Smoking Pledge From Your New Year’s Resolution.

Get My Free Ebook


Post a comment