Enhanced delivery of mda-7/IL-24 using a serotype chimeric adenovirus (Ad.5/3) in combination with the Apogossypol derivative BI-97C1 (Sabutoclax) improves therapeutic efficacy in low CAR colorectal cancer cells

Abstract

Adenovirus (Ad)-based gene therapy represents a potentially viable strategy for treating colorectal cancer. The infectivity of serotype 5 adenovirus (Ad.5), routinely used as a transgene delivery vector, is dependent on Coxsackie-adenovirus receptors (CAR). CAR expression is downregulated in many cancers thus preventing optimum therapeutic efficiency of Ad.5-based therapies. To overcome the low CAR problem, a serotype chimerism approach was used to generate a recombinant Ad (Ad.5/3) that is capable of infecting cancer cells via Ad.3 receptors in a CAR-independent manner. We evaluated the improved transgene delivery and efficacy of Ad.5/3 recombinant virus expressing melanoma differentiation associated gene-7/interleukin-24 (mda-7/IL-24), an effective wide-spectrum cancer-selective therapeutic. In low CAR human colorectal cancer cells RKO, wild-type Ad.5 virus expressing mda-7/IL-24 (Ad.5-mda-7) failed to infect efficiently resulting in lack of expression of MDA-7/IL-24 or induction of apoptosis. However, a recombinant Ad.5/3 virus expressing mda-7/IL-24 (Ad.5/3-mda-7) efficiently infected RKO cells resulting in higher MDA-7/IL-24 expression and inhibition of cell growth both in vitro and in nude mice xenograft models. Addition of the novel Bcl-2 family pharmacological inhibitor Apogossypol derivative BI-97C1 (Sabutoclax) significantly augmented the efficacy of Ad.5/3-mda-7. A combination regimen of suboptimal doses of Ad.5/3-mda-7 and BI-97C1 profoundly enhanced cytotoxicity in RKO cells both in vitro and in vivo. Considering the fact that Ad.5-mda-7 has demonstrated significant objective responses in a Phase I clinical trial for advanced solid tumors, Ad.5/3-mda-7 alone or in combination with BI-97C1 would be predicted to exert significantly improved therapeutic efficacy in colorectal cancer patients.

Figure 1. Enhanced infectivity of tropism-modified adenovirus (Ad.5/3) in RKO low CAR colorectal cancer cells

A) Coxsackie-adenovirus receptors (CARs) expression on the surface of different colorectal cancer cell lines, the K-S value of RKO is only 3%, whereas it reaches 83% for HCT116. B) HCT116 and RKO cells were infected with Ad.5-Luc and Ad.5/3-Luc at the indicated doses; and luciferase activity was measured after 48 hr. Data represent luciferase activity of Ad.5/3-Luc divided by that of Ad.5-Luc. C) HCT116 and RKO cells were infected with the indicated doses of virus and cell viability was quantified using MTT assay after 3 and 6 days of infection.

Figure 2. Ad.5/3-mda-7 induces MDA-7/IL-24 expression and enhances killing in low CAR expressing RKO cancer cells

A and B. HCT116 and RKO cells were infected with the indicated doses of Ad.5-mda-7 or Ad.5/3-mda-7 and respective controls. After 48 hr of infection total proteins were isolated. The expression of MDA-7/IL-24, activation of PARP (as a marker for apoptosis) and expression of EF1α (as a loading control) were analyzed by Western blotting analyses in RKO (A) and HCT 116 (B) cells. C and D. Percentage of dead cells detected by Trypan blue dye exclusion staining for RKO (C) and HCT 116 (D) cells. Results are mean ± S.D. (n=3).

Figure 3. Ad.5/3-mda-7 induces killing, apoptosis and ER stress in RKO low CAR colorectal cancer cells

RKO Cells were treated with the indicated Ad for two days. A) Histogram of different groups assayed by flow cytometry following Annexin V-FITC/PI staining. B) Graph showing percentage of Annexin V- positive cells. Results are the mean ± S.D. (n=3). C) Changes in GRP94, BiP/GRP78, Bcl-2, MDA-7/IL-24 and EF1α were evaluated by Western blotting analysis.

Figure 4. Anti-tumor effect of Ad.5-mda-7 and Ad.5/3-mda-7 in RKO xenografts in nude mice

Subcutaneous tumor xenografts from RKO cells were established in athymic nude mice; tumors were injected with the indicated Ads over a 4-week period (total of nine injections). A) Measurements of RKO xenograft tumor volumes; points, average (with a minimum of five mice in each group); bars, ± S.D. B) measurement of tumor weights at the end of the study. Bars, ± S.D. C) Tumor images at the end of the study.

Figure 5. Ad.5/3-mda-7 shows enhanced MDA-7/IL-24 production and inhibition of proliferation and angiogenesis in RKO low CAR colorectal cancer cells

Subcutaneous tumor xenografts from RKO cells were established in athymic nude mice; the tumors were injected with the indicated Ads over a 4-week period (total of nine injections). At the end of the experiment the tumors were harvested and formalin-fixed, paraffin-embedded, sectioned and were subjected to further analysis A) Immunohistochemical analysis for MDA-7/IL-24 expression. B) Immunohistochemical analysis for CD31 expression. C) Monitoring of H&E stained sections for cellular density.

Figure 6. Suboptimal doses of tropism-modified adenoviruses (Ad.5/3) and BI-97C1 (Sabutoclax) synergize resulting in decreased viability of RKO low CAR colorectal cancer cells

A) RKO cells were treated with the indicated concentrations of BI-97C1 (Sabutoclax). After 3 and 6 days, cell viability was evaluated by MTT assays. Bars, ± S.D. (n = 3). B) RKO cells were infected with 50 pfu/cell of Ad.5/3-vec or Ad.5/3-mda-7 for 24 hr after which cells were treated with indicated concentrations BI-79C1 (Sabutoclax) and MTT assay was performed at 3 and 6 days. C) Analysis of expression of the indicated proteins by Western blotting.

Figure 7. The combination of Ad.5/3-mda-7 and BI-97C1 (Sabutoclax) additively inhibit the growth of low-CAR colorectal cancer xenografts in vivo

Subcutaneous tumor xenografts from RKO cells were established in athymic nude mice; the tumors were injected with the indicated Ads with and without BI-97C1 (Sabutoclax) over a 4-week period (total of nine injections each). A) Measurements of RKO xenograft tumor volumes; points, average (five mice in each group); bars, ± S.D. B) Images of the tumors at the end of the study. C) Measurement of tumor weight at the end of the study; columns, mean; bars, ± S.D.