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Lung cancers express non-neuronal, cholinergic autoparacrine loop, which facilitates tumor growth. Interruption of M3 muscarinic cholinergic signaling has been reported to inhibit small cell lung cancer (SCLC) growth. The purpose of this study is to investigate if blocking autoparacrine muscarinic cholinergic signaling could inhibit non-small cell lung cancer (NSCLC) growth and possible underlying mechanisms. Our results showed that PC9 and A549 cells expressed all 5 subtypes of muscarinic receptor (mAChR) and blocking M2 mAChR (M2R) signaling using selective antagonist methoctramine or short hairpin RNA (shRNA) inhibited tumor cell proliferation in vitro and in vivo. Consistent with AChR agonists stimulating p44/42 MAPK (Erk1/2) and Akt phosphorylation, blocking M2R signaling decreased MAPK and Akt phosphorylation, indicating that non-neuronal ACh functions as an autoparacrine growth factor signaling in part through activation of M2R and downstream MAPK and Akt pathways. Importantly, further studies revealed that blocking M2R signaling also reversed epithelial-mesenchymal transition (EMT) in vitro and in vivo, indicating that non-neuronal ACh promotes EMT partially through activation of M2R. These findings demonstrate that M2R plays a role in the growth and progression of NSCLC and suggest M2R antagonists may be an efficacious adjuvant therapy for NSCLC.

Cancer gene therapy requires tumor-specific delivery and expression of a transgene to maximize antitumor efficacy and minimize side effects. In this study, we developed a new tumor-targeting, homologous recombination-based adenovirus vector system, HRAVS. HRAVS is composed of two adenovirus vectors, Ad.CMV.IR containing reverse sequence (IR) and a CMV promoter and Ad.IR.EGFP comprising the report gene EGFP and IR. For improved viral DNA replication and transgene expression, the E1a gene was added to HRAVS to generate the enhanced HRAVS, EHRAVS, which consists of Ad.CMV.IR and Ad.IR.EGFP/E1a. The optimal vector composition ratio of Ad.CMV.IR to Ad.IR.EGFP or Ad.IR.EGFP/E1a was identified as 30:70 based on EGFP expression efficiency in tumor cells. The transgene expression of HRAVS and EHRAVS was efficiently and specifically activated in tumor cells only and not in normal cells. Moreover, compared with HRAVS, EHRAVS infection led to higher virus yields and transgene expression and higher toxicity to tumor cells, and these results could be related to the involvement of E1a genes. The results in present study suggest the need for in vivo antitumor study using these new dual-Ad vector systems based on the homologous recombination.

Virotherapy employing conditionally replicative adenovirus (CRAd) represents a novel targeted strategy for the hepatocellular carcinoma (HCC) treatment. In this study, we explored the potential influence of E3 region, which encodes several TRAIL-inhibiting proteins (E3-6.7K, E3-10.4K/14.5K and E3-14.7K), on CRAd mediated cytotoxicity to HCC cells. Two E1B-55 kDa-deleted CRAds containing E3 region (Ad.DeltaE1B) or no E3 region (Ad.DeltaE1B.DeltaE3) were fabricated. Ad.DeltaE1B.DeltaE3 exhibited higher cytocidal potency than Ad.DeltaE1B in all tested HCC cells (Hep3B, BEL-7404, BEL-7402, HuH7, PLC/PRF/5 and HepG2), suggesting that Ad.DeltaE1B.DeltaE3 mediated cytotoxicity was partly attributed to the absence of E3 region encoding TRAIL-inhibiting proteins. In representative Hep3B cells, Ad.DeltaE1B.DeltaE3 led to more drop of mitochondrial membrane potential (MMP) and much lower ATP level than Ad.DeltaE1B. Moreover, Ad.DeltaE1B.DeltaE3 induced early apoptotic cells and the late apoptotic/necrotic cells for three and four times more than those infected by Ad.DeltaE1B. The cytotoxicity to all TRAIL endogenously expressing HCC cells and MMP drop of Hep3B cells induced by Ad.DeltaE1B.DeltaE3 but not Ad.DeltaE1B could be significantly inhibited by z-vad-fmk, a pan caspase inhibitor, suggesting that the endogenous TRAIL-mediated apoptotic pathway may be implicated in the cytocidal potency of Ad.DeltaE1B.DeltaE3 on HCC cells although other unknown mechanisms may be also involved. Our findings provided the first evidence that CRAd without E3 region might be a smart choice for the virotherapy of HCC.

The use of immunostimulatory molecule genes aiming at enhancing anti-tumor immunity has emerged as a new approach to treat cancers. 4-1BB signaling, an important costimulatory pathway delivering a signal for T cell activation, survival and growth, has become one of the most promising targets for cancer immunotherapy. In this work, a recombinant nonreplicative adenovirus (Ad.4-1BB scFv) carrying a single-chain Fv fragments (scFv) specific for 4-1BB gene (anti-4-1BB scFv) was generated, haracterized and explored for its stimulation of anti-tumor immunity in immunocompetent C57BL/6 mice. Ad.4-1BB scFv could efficiently infect murine hepatoma Hepa 1-6 cells and induce anti-4-1BB scFv expression on the cell surface. Moreover, Ad.4-1BB scFv did not cause obvious cytotoxicity effect on human and murine tumor cell lines (A549, PLC/PRF/5, Hepa 1-6 and TC-1) even at a high MOI, which suggested Ad.4-1BB scFv had no direct effect on tumor cells. Intratumoral injection of Ad.4-1BB scFv to established Hepa 1-6 tumors significantly suppressed the tumor growth in C57BL/6 mice. The anti-tumor effect might be mainly attributed to the anti-4-1BB scFv-mediated immune activity, as evidenced by enhanced interferon-gamma-producing splenic cells and increased lymphocytes infiltration in the tumor microenvironment. These results indicated that nonreplicative adenovirus carrying the anti-4-1BB scFv gene possessed powerful in vivo anti-tumor efficacy and might be a valuable tool for cancer immunotherapy.

Heat shock proteins (HSPs) possess potent antitumor ability to stimulate immune response. We postulated that intratumor injection of oncolytic adenovirus over-expressing HSPs might be able to exert antitumor activity by inducing antitumor immune response in immunocompetent hosts in addition to the oncolytic activity. In this study, two recombinant oncolytic adenoviruses, Ad.CMV.HSP.IRES.E1a and Ad.CMV.IRES.E1a, were constructed with or without the HSP expression cassette. The HSP expression and cytopathic killing effect in mouse B16 melanoma and human PLC/PRF/5 hepatoma cells were measured after in vitro infection of adenoviruses. Survival rate of immunocompetent C57/BL mice was observed following intratumor injection of recombinant adenoviruses in B16 melanoma xenograft models. To detect the evidence of immune responses, hematoxylin and eosin staining and RT-PCR for IFN-gamma expression in tumor tissues were performed. The Ad.CMV.HSP.IRES.E1a induced significantly higher HSP expression level than Ad.CMV.IRES.E1a in both B16 and PLC/ PRF/5 cells. The cytocidal efficacy of Ad.CMV.HSP.IRES.E1a and Ad.CMV.IRES.E1a in PLC/PRF/5 cells was much higher than that in B16 cells, where the two adenoviruses showed similarly very weak oncolytic effect in vitro. However, Ad.CMV.HSP.IRES. E1a improved animal survival rate and exhibited more potent anti-tumor efficiency than Ad.CMV.IRES.E1a in B16 xenograft models. The enhanced efficacy might be mainly attributed to the HSP-mediated immune activity, as evidenced by the up-regulated expression of IFN-gamma and local heavier intratumor inflammatory cell infiltration. These results indicated that the recombinant oncolytic adenovirus over-expressing HSPs possessed powerful in vivo anti-tumor efficacy and might be a valuable approach for cancer immune gene therapy.