Diluted povidone-iodine inhibits tumor growth through apoptosis-induction and suppression of SOD activity.

Povidone-iodine (PVP-I) is widely used in clinical practice as an antiseptic and flushing agent after surgery to remove a tumor. Our present study was designed to determine whether diluted PVP-I is cytotoxic to colon cancer cells and ascetic tumor cells in vitro and to examine its antitumor effects in vivo. In vitro, CT26 and H22 cells treated with different concentrations of diluted PVP-I (0-1.56 µg/ml) were analyzed using the mononuclear cell direct cytotoxicity assay (MTT) and a flow cytometry assay. In vivo, Balb/c mice injected in the abdominal cavity with CT26 cells or H22 cells were treated intraperitoneally with different concentrations of PVP-I (0-312.5 µg/mouse), cisplatin (40 mg/kg) or 5'-FU (30 mg/kg) or left untreated. In vitro, the studies demonstrated the antiproliferative and significant apoptosis-inducing effects of PVP-I in a dose- and time-dependent manner. In vivo, PVP-I significantly repressed the growth of H22 and CT26 cells in Balb/c mice compared to controls. To explore the mechanism of the antitumor effect of PVP-I, the superoxide dismutase (SOD) activity of ascites extracted from a mouse model and the supernatant of CT26 cells was detected by an SOD kit. The activity of SOD was significantly inhibited in the experimental groups. Taken together, our data suggest that PVP-I exhibits a strong inhibitory effect on tumor growth in colon cancer (CT26) and hepatoma (H22) resulting from apoptosis, both in vitro and in vivo, suggesting a new potential therapeutic approach after tumor excision surgery to colon cancer and hepatoma.

Antitumor effect of mSurvivinThr34→Ala in murine colon carcinoma when administered intravenously.

Colorectal cancer is one of the most common cancers. Survivin is strongly immunogenic in a fraction of colorectal cancer patients. The present study was designed to determine whether full-length mouse Survivin dominant-negative mutant SurvivinT34A has the antitumor activity in a murine colon carcinoma model. The complex of cationic liposome (DOTAP/Chol) to plasmid pORF9-mSurvivin T34A was administered intravenously in a mouse subcutaneous (S. C.) CT 26 tumor model. Apoptotic cells and anti-angiogenesis were evaluated by fluorescent in situ TUNEL assay and by immunohistochemistry with an antibody reactive to CD31, respectively. A 4 h 51Cr release assay was performed to determine Survivin-specific cytotoxicity. The adoptive transfer of CD8+ or CD4+ T-lymphocytes assay was to further explore the roles of immune cell subsets. We demonstrated the complex of cationic liposome (DOTAP/Chol) to plasmid pORF9--mSurvivin T34A when administered intravenously induced an efficient antitumor activity in a S. C. CT26 tumor model in mice. The main mechanism is involved in three aspects: triggering the apoptosis of tumor cells, inhibiting angiogenesis, and inducing Survivin-specific immune response. Our observations may have potential implications for the further exploration of the treatment of human colorectal cancer by intravenous delivery of dominant-negative mutant Survivin T34A.

[Screening and identification of mesenchymal stem cell strains to secret mouse interleukin-12 mediated with lenti-viral vector].

OBJECTIVE: To screen the stable expression cell strains of mouse interleukin-12 (mIL-12) from mouse Mesenchymal Stem Cells (mMSCs) transfected with lenti-mIL-12 virus. METHODS: The mIL-12 cDNA was amplified from plasmid pORF-mIL-12 (Invivogen) by PCR. The cDNA was subcloned into pENTR 11 to generate recombinant plasmid pENTR-mIL-12. Then, pENTR-mIL-12 was homologously recombinated with pLenti6/V5-Dest. The recombinant was named as pLenti6/V5-mIL-12 and confirmed by PCR and DNA sequencing. The Lenti6/V5-mIL-12 virus was packaged using 293FT cells. The Lenti-mIL-12-MSC monoclone was picked from the mMSCs infected by the Lenti6/V5-mIL-12 virus using Blasticidin and verified by RT-PCR and ELISA. RESULTS: The recombinant pLenti6/V5-mIL-12 was constructed. The sequence of amplified mIL-12 gene was consistent with that reported in GenBank. By RT-PCR and ELISA, it was confirmed that the mIL-12 protein could be expressed and secreted into the supernatant of MSC strain culture. CONCLUSION: The recombinant mMSC strains lentivirally engineered to secret mIL-12 were obtained.

[Apoptosis of colon carcinoma cells of mice induced by vesicular stomatitis virus matrix protein in vitro].

OBJECTIVE: To test the inhibitive effect of the matrix protein of vesicular stomatitis virus (VSV) on the proliferation of colon carcinoma cells of mice in vitro. METHODS: The plasmid pcDNA3. 1-M encoding M protein of vesicular stomatitis viruses was transfected with lipofectamine 2000 into the colon carcinoma (CT26) cells of mice. The expression of VSV-M protein in the CT26 cells was detected by Western blot. The effect of vesicular stomatitis virus (VSV) matrix protein on the proliferation and apoptosis of colon carcinoma cells was measured by MTT, PI stainning and flow cytometry. RESULTS: The plasmid expressed M protein in the CT26 cells. Cytopathic effect was shown in the CT26 cells with transfected pcDNA3.1-M. The proliferation of the CT26 cells was suppressed significantly in vitro (76.4%, P < 0.05). The M protein induced apoptosis of the CT26 cells (80.2%), which was higher than'that of the controls (P < 0.05). CONCLUSION: The eukaryotic expression plasmid pcDNA3.1-M encoding M protein of vesicular stomatitis viruses induces apoptosis of CT26 cells, which has implications on the treatment of human colon cancer.

A promising cancer gene therapy agent based on the matrix protein of vesicular stomatitis virus.

The matrix (M) protein of vesicular stomatitis virus (VSV) plays a key role in inducing cell apoptosis during infection. To investigate whether M protein-mediated apoptosis could be used in cancer therapy, its cDNA was amplified and cloned into eukaryotic expression vector pcDNA3.1(+). The recombinant plasmid or the control empty plasmid pcDNA3.1(+) was mixed with cationic liposome and introduced into various tumor cell lines in vitro, including lung cancer cell LLC, A549, colon cancer cell CT26 and fibrosarcoma cell MethA. Our data showed that the M protein induced remarkable apoptosis of cancer cells in vitro compared with controls. Fifty micrograms of plasmid in a complex with 250 microg cationic liposome was injected intratumorally into mice bearing LLC or MethA tumor model every 3 days for 6 times. It was found that the tumors treated with M protein plasmid grew much more slowly, and the survival of the mice was significantly prolonged compared with the mice treated with the control plasmid. In MethA fibrosarcoma, the tumors treated with M protein plasmid were even completely regressed, and the mice acquired longtime protection against the same tumor cell in rechallenge experiments. Both apoptotic cells and CD8(+) T cells were widely distributed in M protein plasmid-treated tumor tissue. Activated cytotoxic T lymphocytes (CTLs) were further detected by means of (51)Cr release assay in the spleen of the treated mice. These results showed that M protein of VSV can act as both apoptosis inducer and immune response initiator, which may account for its extraordinary antitumor effect and warrant its further development in cancer gene therapy.

Vesicular stomatitis virus matrix protein gene enhances the antitumor effects of radiation via induction of apoptosis.

Vesicular stomatitis virus (VSV) matrix (M) protein can directly induce apoptosis by inhibiting host gene expression when it is expressed in the absence of other viral components. Previously, we found that the M protein gene complexed to DOTAP-cholesterol liposome (Lip-MP) can suppress malignant tumor growth in vitro and in vivo; however, little is known regarding the biological effect of Lip-MP combined with radiation. The present study was designed to determine whether Lip-MP could enhance the antitumor activity of radiation. LLC cells treated with a combination of Lip-MP and radiation displayed apparently increased apoptosis compared with those treated with Lip-MP or radiation alone. Mice bearing LLC or Meth A tumors were treated with intratumoral or intravenous injections of Lip-MP and radiation. The combined treatment significantly reduced mean tumor volumes compared with either treatment alone in both tumor models and prolonged the survival time in Meth A tumor models and the intravenous injection group of LLC tumor models. Moreover, the antitumor effects of Lip-MP combined with radiation were greater than their additive effects when compared with the expected effects of the combined treatment in vivo. This study suggests that Lip-MP enhanced the antitumor activity of radiation by increasing the induction of apoptosis.

Induction of apoptosis by phenylisocyanate derivative of quercetin: involvement of heat shock protein.

Quercetin, a widely distributed bioflavonoid, inhibits the growth of various tumor cells. The present study was designed to investigate whether a novel quercetin derivative [phenylisocyanate of quercetin (PHICNQ)] exerts antitumor activity against K562 and CT26 tumor cell lines by inducing apoptosis, and to examine the possible mechanism in the phenomenon. The cell proliferation assay of K562 and CT26 tumor cells was determined by the trypan blue dye exclusion test. Apoptosis of PHICNQ-treated cells was determined by morphological analysis, agarose gel DNA electrophoresis and quantitated by flow cytometry after staining with propidium iodide. Cell cycle was evaluated by flow cytometry. The expression of heat shock protein 70 was checked by Western blot analysis. Our results showed that PHICNQ inhibited the proliferation of K562 and CT26 cells in a dose-dependent and time-dependent manner. PHICNQ was 308- and 73-fold more active on CT26 and K562 cells than quercetin, respectively. In addition to this cytostatic effect, treatment of K562 and CT26 tumor cells with PHICNQ induced apoptosis. PHICNQ treatment downregulated the expression of heat shock protein 70 more dramatically than quercetin treatment. These results suggest that PHICNQ is a more powerful antiproliferative derivative than quercetin, with cytostatic and apoptotic effects on K562 and CT26 tumor cells. PHICNQ may trigger apoptosis in tumor cells through inhibition of heat shock protein 70 synthesis and expression.

[Effects of vesicular stomatitis virus matrix protein on proliferation and apoptosis of mouse LL/2c cells].

BACKGROUND & OBJECTIVE: Vesicular stomatitis virus (VSV), an oncolytic virus, is an attractive candidate for tumor therapy. Although previous studies showed obvious antitumor effects of VSV on human A549 and mouse LL/2c tumor models, the clinical application of a live virus is confronted with the problem of bio-safety. The matrix (M) protein of VSV is related to the antitumor effect of VSV. This study was to investigate the effects of VSV-M protein on the proliferation and apoptosis of mouse LL/2c tumor cells. METHODS: A eukaryotic expression plasmid pcDNA3.1-M encoding VSV-M protein was constructed by molecular cloning technique, and analyzed by enzyme digestion, polymerase chain reaction (PCR), and DNA sequencing, then transfected into LL/2c cells. The expression of VSV-M protein in LL/2c cells was detected by reverse transcription-polymerase chain reaction (RT-PCR) and Western blot. The effect of VSV-M protein on proliferation of LL/2c cells was assessed by MTT assay; its effect on cell apoptosis was assessed by DNA ladder and Hoechst 33528 staining. RESULTS: A plasmid pcDNA3.1-M encoding VSV-M protein was constructed successfully and identified. After transfection, the expression of VSV-M protein was detected in LL/2c cells, morphologic changes of LL/2c cells was observed under microscope, inhibition rate of cell survival was 41.3% (P<0.05), DNA ladder was detected, apoptotic nuclei was observed. CONCLUSION: VSV-M transfection could inhibit proliferation and induce apoptosis of LL/2c cells.

[Construction of mammalian co-expression plasmid pIRES -LMP1-HSP90 beta and its expression in vitro].

OBJECTIVE: This study sought to clone Epstein-Barr virus latent membrane protein 1 (LMP1) and heat shock protein 90 beta (HSP90 beta) of nasopharyngeal carcinoma (NPC), to construct the mammalian co-expression plasmid pIRES-LMP1-HSP90 beta, and to detect the expression of the plasmid in vitro. METHODS: Total RNA was isolated from human NPC by cloning technique, their cDNA fragments of LMP1 gene and HSP90 beta gene were gained by RT-PCR, and their cDNA fragments were constructed into the mammalian co-expression plasmid vector pIRES. The inserted target genes in the mammalian co-expression plasmid were verified by nucleotide sequencing. COS cell line was transfected with this mammalian co-expression plasmid using lipofectin reagent. The expression of LMP1 and HSP90 beta molecules were detected by RT-PCR and Western blot technique. RESULTS: The mammalian expression plasmid pIRES-LMP1-HSP90 beta was obtained by cloning technique. The nucleotide sequences of LMP1 gene and HSP90 beta gene in this mammalian co-expression plasmid had high homology with EBV-LMP1 (100%) and human HSP90 beta (100%) respectively. After transfection with this mammalian co-expression plasmid, the LMP1 and HSP90 beta molecules were expressed in COS cells. CONCLUSION: The constructed mammalian co-expression plasmid pIRES-LMP1-HSP90 beta can express LMP1 and HSP90 beta molecules in vitro at the same time.