Indole-3-carbinol synergistically sensitises ovarian cancer cells to bortezomib treatment.

BACKGROUND: Bortezomib is a proteasome inhibitor with minimal clinical activity as a monotherapy in solid tumours, but its combination with other targeted therapies is being actively investigated as a way to increase its anticarcinogenic properties. Here, we evaluate the therapeutic potential of co-treatment with bortezomib and indole-3-carbinol (I3C), a natural compound found in cruciferous vegetables, in human ovarian cancer. METHODS: We examined the effects of I3C, bortezomib and cisplatin in several human ovarian cancer cell lines. Synergy was determined using proliferation assays and isobologram analysis. Cell cycle and apoptotic effects were assessed by flow cytometry. The mechanism of I3C and bortezomib action was determined by RNA microarray studies, quantitative RT-PCR and western blotting. Antitumour activity of I3C and bortezomib was evaluated using an OVCAR5 xenograft mouse model. RESULTS: I3C sensitised ovarian cancer cell lines to bortezomib treatment through potent synergistic mechanisms. Combination treatment with bortezomib and I3C led to profound cell cycle arrest and apoptosis as well as disruptions to multiple pathways, including those regulating endoplasmic reticulum stress, cytoskeleton, chemoresistance and carcinogen metabolism. Moreover, I3C and bortezomib co-treatment sensitised ovarian cancer cells to the standard chemotherapeutic agents, cisplatin and carboplatin. Importantly, in vivo studies demonstrated that co-treatment with I3C and bortezomib significantly inhibited tumour growth and reduced tumour weight compared with either drug alone. CONCLUSION: Together, these data provide a novel rationale for the clinical application of I3C and bortezomib in the treatment of ovarian cancer.

Typical and atypical trafficking pathways of Ad5 penton base recombinant protein: implications for gene transfer.

The adenovirus (Ad) penton base protein facilitates viral infection by binding cell surface integrins, triggering receptor-mediated endocytosis and mediating endosomal penetration. Given these multiple functions, recombinant penton base proteins have been utilized as non-viral vehicles for gene transfer by our lab and others. Although we have previously demonstrated that penton base-derived vectors undergo integrin-specific binding and cell entry, less than desirable levels of gene expression have led us to re-evaluate the recombinant penton base as an agent for gene delivery. To do so, we have examined here the intracellular trafficking of an Ad serotype 5 (Ad5) recombinant penton base protein (PB). Here, we not only observed that PB utilizes a similar, typical trafficking pathway of whole Ad, but also found that PB entered HeLa cells through pathways not yet identified as contributing to cell entry by the whole virus. We show by high-resolution confocal microscopy and biochemical methods that binding to alphav-integrins is a requirement for cell entry, but that early internalization stages did not substantially pass through clathrin-positive and early endosomal compartments. Moreover, a subpopulation of internalized protein localized with caveolin-positive compartments and Golgi markers, suggesting that a certain percentage of proteins pass through non-clathrin-mediated pathways. Similar to the virus, trafficking toward the nucleus was affected by disruption of microtubules and dynein. The majority of penton base molecules avoided the lysosome while facilitating early vesicle release of low molecular weight dextran molecules. In further support of a vesicle escape capacity, a subpopulation of internalized penton base appeared to enter the nucleus, as observed by high-resolution confocal microscopy and cell fractionation. As a confirmation of these findings, we demonstrate that a recombinant penton base facilitated cytosolic entry of an siRNA molecule as observed by RNA interference of a marker gene. Based on our findings here, we suggest that whereas soluble penton base proteins may enter cells through clathrin- and non-clathrin-mediated pathways, vesicle escape and nuclear delivery appear to be supported by a clathrin-mediated pathway. As our previous efforts have focused on utilizing recombinant penton base proteins as delivery agents for therapeutics, these findings allow us to evaluate the use of the penton base as a cell entry and intracellular trafficking agent, and may be of interest concerning the development of vectors for efficient delivery of therapeutics to cells.

The Ad5 fiber mediates nonviral gene transfer in the absence of the whole virus, utilizing a novel cell entry pathway.

The interesting discovery reported here that soluble adenovirus serotype 5 (Ad5) fiber proteins enter cells without the virus was a serendipitous result during our development of Ad5 capsid proteins as nonviral gene transfer vectors. The Ad5 capsid fiber and penton proteins mediate infection. The fiber docks to a noninternalizing cell surface protein called the coxsackievirus-Ad receptor (CAR), followed by penton binding to integrins, triggering integrin-mediated endocytosis of the virus. In our previous work, we assembled the nonviral complex, 3PO, which utilized the penton to mediate gene transfer through integrin binding and endocytosis. Here, we tested whether incorporating the fiber targets 3PO to CAR, thus recapitulating the Ad5 infection pathway. As CAR is not an endocytic receptor, we were surprised to find that the fiber alone, without the penton, enabled gene transfer by binding CAR, but internalizing through an unknown mechanism. We show here that the fiber distributes to the nucleus and cytoplasm after temperature-independent uptake, whereas the penton accumulates around the nucleus after temperature-dependent uptake. Fiber uptake by HeLa cells is also actin-dependent, requires the fiber tail/shaft region, and is largely inhibited by heparin. This study raises the possibility that alternative pathways may enable both viral and nonviral cell entry.