Xin Cao

Potent Antitumor Effect of Interleukin-24 Gene in the Survivin Promoter and Retinoblastoma Double-Regulated Oncolytic Adenovirus

Interleukin (IL)-24 is an excellent therapeutic gene for cancer therapy. In this work, IL-24 was inserted into Ad.sp-E1A(Delta24), an oncolytic adenovirus with a 24-bp deletion in the E1A gene, which was driven by the survivin promoter to form Ad.sp-E1A(Delta24)-IL-24. Ad.sp-E1A(Delta24)-IL-24 has an excellent antitumor effect in vitro for human nasopharyngeal, liver, lung, and cervical carcinoma cell lines but does no or little damage to normal cell lines L-02 and WI38. Furthermore, it achieved nearly complete inhibition (although not elimination) of NCI-H460 lung carcinoma growth in nude mice. The antitumor efficacy of Ad.sp-E1A(Delta24)-IL-24 on NCI-H460 cells was clearly mediated by apoptosis, because it induced caspase-3 and poly(ADP-ribose) polymerase cleavage. This is the first report of Ad.sp-E1A(Delta24)-IL-24 with such an excellent, broad, and specific antitumor effect in vitro and nearly complete inhibition of lung tumor growth in vivo.

Augmenting the Antitumor Effect of TRAIL by SOCS3 with Double-Regulated Replicating Oncolytic Adenovirus in Hepatocellular Carcinoma

Aberrant JAK/STAT3 pathway has been reported to be related to hepatocellular carcinoma (HCC) in many cell lines. In this study, a double-regulated oncolytic adenovirus vector that can replicate and induce a cytopathic effect in alpha-fetoprotein (AFP)-positive HCC cell lines with p53 dysfunction was successfully constructed. Two therapeutic genes, suppressor of cytokine signaling 3 (SOCS3) and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), were chosen and incorporated into this vector system, respectively. The combined treatment of AFP-D55-SOCS3 and AFP-D55-TRAIL (2:3 ratio) exhibited potent antitumor activity in AFP-positive HCC cell lines compared with any other treatment both in vitro and in vivo. Specific replication and low progeny yield in AFP-positive HCC cell lines rendered these double-regulated oncolytic adenoviruses remarkably safe. Our data demonstrated that restoration of SOCS3, which inhibits the JAK/STAT3 pathway, by AFP-D55-SOCS3 not only could antagonize HCC therapeutic resistance to TRAIL and adenoviruses, but could also induce cell cycle arrest in HCC cell lines. SOCS3 could down-regulate Cyclin D1 and anti-apoptotic proteins such as XIAP, Survivin, Bcl-xL, and Mcl-1, which are responsible for the synergistic inhibitory effects of AFP-D55-SOCS3 and AFP-D55-TRAIL. Dual gene and double-regulated oncolytic adenoviruses may provide safety and excellent antitumor effects for liver cancer, which is the advantage of a cancer-targeting gene virotherapy strategy.

Potent Antitumor Efficacy of ST13 for Colorectal Cancer Mediated by Oncolytic Adenovirus via Mitochondrial Apoptotic Cell Death

ST13 is a cofactor of heat shock protein 70 (Hsp70). To date, all data since the discovery of ST13 in 1993 until more recent studies in 2007 have proved that ST13 is downregulated in tumors and it was proposed to be a tumor suppressor gene, but no work reported its antitumor effect and apoptotic mechanism. In the work described in this paper, ST13 was inserted into ZD55, an oncolytic adenovirus with the E1B 55-kDa gene deleted, to form ZD55-ST13, which exerts an excellent antitumor effect in vitro and in an animal model of colorectal carcinoma SW620 xenograft. ZD55-ST13 inhibited tumor cells 100-fold more than Ad-ST13 and ZD55-EGFP in vitro. However, ZD55-ST13 showed no damage of normal fibroblast MRC5 cells. In exploring the mechanism of ZD55-ST13 in tumor cell killing, we found that ZD55-ST13-infected SW620 cells formed apoptotic bodies and presented obvious apoptosis phenomena. ZD55-ST13 induced the upregulation of Hsp70, the downregulation of antiapoptotic gene Bcl-2, and the release of cytochrome c. Cytochrome c triggered apoptosis by activating caspase-9 and caspase-3, which cleave the enzyme poly(ADP-ribose) polymerase in ZD55-ST13-infected SW620 cells. In summary, overexpressed ST13 as mediated by oncolytic adenovirus could exert potent antitumor activity via the intrinsic apoptotic pathway and has the potential to become a novel therapeutic for colorectal cancer gene therapy.

A new oncolytic adenoviral vector carrying dual tumour suppressor genes shows potent anti-tumour effect

Cancer Targeting Gene‐Viro‐Therapy (CTGVT) is a promising cancer therapeutical strategy that strengthens the anti‐tumour effect of oncolytic virus by expressing inserted foreign anti‐tumour genes. In this work, we constructed a novel adenoviral vector controlled by the tumour‐specific survivin promoter on the basis of the ZD55 vector, which is an E1B55KD gene deleted vector we previously constructed. Compared with the original ZD55 vector, this new adenoviral vector (ZD55SP/E1A) showed much better ability of replication and reporter gene expression. We then combined anti‐tumour gene interleukine‐24 (IL‐24) with an RNA polymerase III‐dependent U6 promoter driving short hairpin RNA (shRNA) that targets M‐phase phosphoprotein 1 (MPHOSPH1, a newly identified oncogene) by inserting the IL‐24 and the shRNA of MPHOSPH1 (shMPP1) expression cassettes into the new ZD55SP/E1A vector. Our results demonstrated excellent anti‐tumour effect of ZD55SP/E1A‐IL‐24‐shMPP1 in vitro on multiple cancer cell lines such as lung cancer, liver cancer and ovarian caner. At high multiplicity‐of‐infection (MOI), ZD55SP/E1A‐IL‐24‐shMPP1 triggered post‐mitotic apoptosis in cancer cells by inducing prolonged mitotic arrest; while at low MOI, senescence was induced. More importantly, ZD55SP/E1A‐IL‐24‐shMPP1 also showed excellent anti‐tumour effects in vivo on SW620 xenograft nude mice. In conclusion, our strategy of constructing an IL‐24 and shMPP1 dual gene expressing oncolytic adenoviral vector, which is regulated by the survivin promoter and E1B55KD deletion, could be a promising method of cancer gene therapy.

Prostate Cancer-Specific and Potent Antitumor Effect of a DD3-Controlled Oncolytic Virus Harboring the PTEN Gene

Prostate cancer is a major health problem for men in Western societies. Here we report a Prostate Cancer-Specific Targeting Gene-Viro-Therapy (CTGVT-PCa), in which PTEN was inserted into a DD3-controlled oncolytic viral vector (OV) to form Ad.DD3.E1A.E1B(Δ55)-(PTEN) or, briefly, Ad.DD3.D55-PTEN. The woodchuck post-transcriptional element (WPRE) was also introduced at the downstream of the E1A coding sequence, resulting in much higher expression of the E1A gene. DD3 is one of the most prostate cancer-specific genes and has been used as a clinical bio-diagnostic marker. PTEN is frequently inactivated in primary prostate cancers, which is crucial for prostate cancer progression. Therefore, the Ad.DD3.D55-PTEN has prostate cancer specific and potent antitumor effect. The tumor growth rate was almost completely inhibited with the final tumor volume after Ad.DD3.D55-PTEN treatment less than the initial volume at the beginning of Ad.DD3.D55-PTEN treatment, which shows the powerful antitumor effect of Ad.DD3.D55-PTEN on prostate cancer tumor growth. The CTGVT-PCa construct reported here killed all of the prostate cancer cell lines tested, such as DU145, 22RV1 and CL1, but had a reduced or no killing effect on all the non-prostate cancer cell lines tested. The mechanism of action of Ad.DD3.D55-PTEN was due to the induction of apoptosis, as detected by TUNEL assays and flow cytometry. The apoptosis was mediated by mitochondria-dependent and -independent pathways, as determined by caspase assays and mitochondrial membrane potential.

HCCS1-armed, quadruple-regulated oncolytic adenovirus specific for liver cancer as a Cancer Targeting Gene-Viro-Therapy strategy

BackgroundIn previously published studies, oncolytic adenovirus-mediated gene therapy has produced good results in targeting cancer cells. However, safety and efficacy, the two most important aspects in cancer therapy, remain serious challenges. The specific expression or deletion of replication related genes in an adenovirus has been frequently utilized to regulate the cancer cell specificity of a virus. Accordingly, in this study, we deleted 24 bp in E1A (bp924-bp947) and the entirety of E1B, including those genes encoding E1B 55kDa and E1B19kDa. We used the survivin promoter (SP) to control E1A in order to construct a new adenovirus vector named Ad.SP.E1A(Δ24).ΔE1B (briefly Ad.SPDD). HCCS1 (hepatocellular carcinoma suppressor 1) is a novel tumor suppressor gene that is able to specifically induce apoptosis in cancer cells. The expression cassette AFP-HCCS1-WPRE-SV40 was inserted into Ad.SPDD to form Ad.SPDD-HCCS1, enabling us to improve the safety and efficacy of oncolytic-mediated gene therapy for liver cancer.ResultsAd.SPDD showed a decreased viral yield and less toxicity in normal cells but enhanced toxicity in liver cancer cells, compared with the cancer-specific adenovirus ZD55 (E1B55K deletion). Ad.SPDD-HCCS1 exhibited a potent anti-liver-cancer ability and decreased toxicity in vitro. Ad.SPDD-HCCS1 also showed a measurable capacity to inhibit Huh-7 xenograft tumor growth on nude mice. The underlying mechanism of Ad.SPDD-HCCS1-induced liver cancer cell death was found to be via the mitochondrial apoptosis pathway.ConclusionsThese results demonstrate that Ad.SPDD-HCCS1 was able to elicit reduced toxicity and enhanced efficacy both in vitro and in vivo compared to a previously constructed oncolytic adenovirus. Ad.SPDD-HCCS1 could be a promising candidate for liver cancer therapy.

A double-regulated oncolytic adenovirus with improved safety for adenocarcinoma therapy

Safety and efficiency are equally important to be considered in developing oncolytic adenovirus. Previously, we have reported that ZD55, an oncolytic adenovirus with the deletion of E1B-55K gene, exhibited potent antitumor activity. In this study, to improve the safety of ZD55, we utilized MUC1 promoter to replace the native promoter of E1A on the basis of ZD55, and generated a double-regulated adenovirus, named MUD55. Our data demonstrated that the expression of early and late genes of MUD55 was both reduced in MUC1-negative cells, resulting in its stricter glandular-tumor selective progeny production. The cytopathic effect of MUD55 was about 10-fold lower than mono-regulated adenovirus ZD55 or Ad.MUC1 in normal cells and not obviously attenuated in glandular tumor cells. Moreover, MUD55 showed the least liver toxicity when administrated by intravenous injection in nude mice. These results indicate that MUD55 could be a promising candidate for the treatment of adenocarcinoma.

ASK1–JNK signaling cascade mediates Ad-ST13-induced apoptosis in colorectal HCT116 cells†

ST13, a co‐factor of heat shock protein, has shown potential antitumor efficacy for colorectal cancer in our previous study. However, the molecular mechanisms governing ST13‐induced apoptosis are poorly understood. Here, we demonstrate that Ad‐ST13 (ST13 mediated by adenovirus) activates apoptosis signal‐regulated kinase (ASK1) and c‐Jun N‐terminal kinase (JNK) but not p38 (mitogen‐activated protein kinase) in human colorectal HCT116 cells. Ad‐ST13 also increases extracellular‐regulated kinase (ERK) phosphorylation levels, but the change is due to adenovirus replication. Overexpression of ST13 also increases the transcription activity of AP‐1. Blocking ASK1–JNK pathway affects Ad‐ST13‐mediated colorectal cell apoptosis, decreases the release of cytochrome c in cytoplasm and caspase activation. Because ASK1 is known to contain a tetratricopeptide repeat (TPR)‐acceptor site and ST13 has TPR domain, we found the interaction between ST13 and ASK1. These results strongly indicate Ad‐ST13 triggers colorectal cell apoptosis via ASK1–JNK signaling cascade. J. Cell. Biochem. 110: 581–588, 2010.

Cancer Targeting Gene-Viro-Therapy and Its Promising Future: A Trend in Both Cancer Gene Therapy and Cancer Virotherapy

The “Cancer Targeting Gene–Viro–Therapy, CTGVT,” strategy was initiated by us in 1999-2001, which was constructed by inserting an antitumor gene into an oncolytic viral vector (OV), it is actually an OV-gene therapy. The CTGVT (OV-gene) shows much higher antitumor effect in vitro and/or in vivo than that of either respective gene therapy alone or respective oncolytic virus therapy alone. We have been persisted to study this strategy for more than 10 years and about 70 with rather high IF value papers have been published. The antitumor order of drugs is OV-gene>OV≥Ad-gene. The CTGVT (OV-gene) has been recently become a hot topic in the cancer field which can be validated by the facts that (1) Amgen paid 1 billion USD to BioVex Inc. to purchase a CTGVT (OV-gene) product, OncoHSV-GM-CSF 205 (OV from herpes simplex virus); (2) OncoPox-GM-CSF (poxvirus) 206 has been published in Nature 2011, because that OncoPox-GM-CSF could be intravenous administration and targeted to the metastasize tumor. We have constructed many modified CTGVT (OV-gene) drugs with complete eradication the xenografted tumor as shown in the text. It will be sure that we can make drugs with higher antitumor effect than that of the 1 billion US OncoHSV-GM-CSF or the Nature paper’s OncoPox-GM-CSF.

Possibility to Partly Win the War Against Cancer

We have worked out two strong antitumor strategies, namely, two antitumor stars. One is the “Cancer Targeting Gene-Viro-Therapy, CTGVT” strategy and the other is the super interferon (sIFN-I) protein. By the combination of the above two antitumor stars with other biotherapy such as antibodies, CIK (which will be described below) and surgery, chemotherapy, and radiotherapy, we can partly win the war against cancer.