CT26 Cell Lines Research Articles

Cytotoxic Activity of Halogenated Monoterpenes from Plocamium cartilagineum

Nine halogenated monoterpenes isolated from the red alga Plocamium cartilagineum have been evaluated for their cytotoxic effects on the tumor cell lines CT26 (murine colon adenocarcinoma), SW480 (human colon adenocarcinoma), HeLa (human cervical adenocarcinoma) and SkMel28 (human malignant melanoma) with several multidrug resistance mechanisms and the mammalian non-tumor cell line CHO (Chinese hamster ovary cells). The activities of these compounds were compared with those of the insecticide gamma-hexachlorocyclohexane (lindane) due to chemical structure similarities. Compounds 1, 2, 3, and 5 exhibited selective cytotoxicity against colon and cervical adenocarcinoma cells. Interestingly, the effect of compound 3 was specific and irreversible to human colon adenocarcinoma SW480 cells, which overexpress the transmembrane P-glycoprotein often related to chemoresistance. None of the anti-tumor doses of these compounds was cytotoxic against CHO cells. Furthermore, analysis of cellular extracts after incubation with the test compounds and rotenone (positive uptake control) demonstrated the intracellular accumulation of 1, 2, 3, and 5.

270. Treatment of CT26 Tumors with Dendritic Cells Transduced with a Recombinant SV40 Vector Expressing Interleukine 15

Tumors can be treated with cytokines that activate the immuno-response against tumor cells. Viral vectors have been used to direct cytokine expression to the tumor and surrounding tissues or to cells like dendritic cells (DCs). We decided to study the efficiency of recombinant vectors based on Simian Virus 40 virus (SV40) to deliver interleukine 12 (IL12) or interleukine 15 (IL15) to the tumor or to transduced DCs. We produced recombinant viruses expressing firefly luciferase (rSVLuc), murine IL12 (rSVIL12) and murine IL15 (rSVIL15). Expression, secretion and activity of recombinant SV40 expressed IL12 and IL15, was demonstrated by several means. Infection of the tumour cell line CT26 and of human DCs by rSVIL12 was determined by Elisaagainst murine IL12. In vitro pre-treatment of CT26 cells with rSVLuc, rSVIL12 or rSVIL15 before subcutaneous inoculation in BALB/C mice did not prevent tumor formation. Intratumoral treatment of CT26 subcutaneous tumours with rSVLuc, rSVIL12 or rSVIL15 had no effect in tumor growth, even when three intratumoral viral injections were performed. We also used these vectors to infect in vitro differentiated BALB/C mice DCs obtained from bone marrow. Infection of immature DCs with rSV40 vectors did not change the expression of cell surface markers CD80, CD86, CD11c, CD40, HLA-I and HLA-II, indicating that DCs tranduced by rSV40 vectors maintained immature after virus infection. We assayed the antitumoral efficiency of infected and maturated DCs injected intratumoraly or infected immature DCs injected intratumoraly or intravenously. The best result was obtained with immature DCs injected directly inside the tumor. 73% tumour remissions were obtained with DCs expressing IL15 and 40% remissions with DCs transduced by rSVIL12. An elispot and a cytotoxic assay indicated that mice treated with DCs expressing IL15 had the highest amount of INFg producing T cells and cytotoxic T cells. These results can be explained by the ability of IL15 to enhance the interaction between DCs and T cells. We conclude that DCs infected with rSVIL15 vectors could be a good alternative to activate the immuno-response against tumor cells.

Establishment and characterization of a rectal cancer model in mice: application to cytokine gene therapy.

We established an orthotopic animal model of rectal cancer in mice and applied this model to the study of the antitumor effects of cytokine-assisted tumor vaccine. The CT-26 murine colon adenocarcinoma cells were inoculated into the submucosa of the rectum of the mice to induce the rectal tumor. The tumor growth rate and the survival time of the mice were observed. The cDNA of granulocyte-macrophage colony-stimulating factor (GM-CSF) was transduced to the CT-26 cell line via a retroviral vector, and the therapeutic effects of irradiated GM-CSF secreting tumor vaccine on the rectal tumor were investigated. All the mice implanted with the wild-type tumor cells had tumor growth in the rectum and died. The mean survival time of the mice was 28.9 days. Two doses of irradiated GM-CSF secreting tumor vaccine administered on days 0 and 3 after tumor cell implantation significantly prolonged the survival of the mice with rectal tumor compared with that of the control groups ( P<0.0001). In contrast, no antitumor effect was observed when the treatment with GM-CSF secreting tumor vaccine was delayed to 3 days after tumor cell implantation ( P>0.17). The results suggest that cytokine gene therapy exerts an antitumor effect on small tumors and may be considered as an adjuvant immunotherapy of rectal cancers and prevention of reimplantation of tumor cells disseminated during or following surgery. The orthotopic animal model of the rectal cancer in mice could be applied to the in vivo experimental studies of rectal cancer.

Effects of anesthesia-induced modest hypothermia on cellular radiation sensitivity

To assess the mechanisms of modest hypothermia (MH) and its effects on cellular radiation response, a model of anesthesia-induced modest hypothermia (AIMH) in the adult mice and a model of pure MH in the newborn mice were established. The survival rate of lethally irradiated mice was increased to 72% through AIMH before irradiation. Both apoptosis and necrosis of human fetal bone marrow CD34(+) hematopoietic stem cells cultured under MH were significantly decreased as detected by MTT and flow cytometry, with three-color labeled by PE-CD(34) (+)/ FITC-AnnexinV /7AAD. The survival and proliferation of mouse bone marrow MNC treated with MH after irradiation were also increased. The MH exerted similar protective effects on the leukemia cell lines A20, HL60, K562 to the normal bone marrow cells, but it enhanced the radiation sensitivity of leukemia cell line FBL3 and mouse melanoma B16F10. No effects have been found on the radiation sensitivity of those cells treated with MH before irradiation. The results also showed that MH mediated the effects on radiation sensitivity, in addition to increasing the oxygen tension. These results show different effects of MH on different cells: (i) AIMH exerts a protective effect on the normal hematopoietic stem cells, some leukemia cell lines A20, HL60, K562, and some neoplasma 3LL, LOVO. And MH exhibits a synthetic effect with anesthetic. (ii) MH enhances the radiation sensitivity of another leukemia and neoplasma cell lines FBL3, B16F10 and CT26. Therefore, AIMH has a potential to enhance the effects of radiation-therapy and decrease side effects on some tumors.

Ablation of Tumor Cells In Vivo by Direct Injection of HSV‐Thymidine Kinase Retroviral Vector and Ganciclovir Therapy

The introduction of therapeutic genes into proliferating tumor cells in vivo by direct intralesional injection of retroviral vectors can provide an effective and valuable approach for the treatment of a variety of solid tumor types. Efficient transduction of tumor cells in situ by direct injection was demonstrated using a retroviral vector containing the beta-galactosidase (beta-gal) gene. Ablation therapy in vivo was demonstrated using a retroviral vector containing the Herpes simplex virus thymidine kinase gene (HSV-TK) to deliver the TK gene into the murine colorectal tumor cell line CT26. Ablation of CT26 tumor cells in situ was achieved by directly injecting high-titer HSV-TK retroviral vector preparations into the site of tumor cell inoculation followed by intraperitoneal (i.p.) delivery of ganciclovir (GCV). This gene therapy strategy demonstrated a markedly lower rate of tumor progression, with several complete regressions, compared to animals in control groups. We also demonstrated that resistance to subsequent challenges with unmodified CT26 cells and an enhanced cellular immune response is associated with tumor regression in immunocompetent animals. Our results demonstrate the feasibility of direct in situ administration of HSV-TK retroviral vectors for the treatment of cancer and suggest that a cellular immune response may be elicited by this therapy.

Vaccination with Dendritic Cells Inhibits the Growth of Hepatic Metastases in B6 Mice

Dendritic cells (DC) are specialized antigen-presenting cells that can activate naive and mature T-cells, induce cellular immunity, and stimulate strong antitumor reactionsin vivo.This study was undertaken to examine the function of DC vaccines in suppressing the growth of hepatic metastases in C57BL/6 mice. Experimental mice received two iv doses of 1 × 106bone marrow-derived DC, either unpulsed or pulsed with MCA-106 fibrosarcoma cell lysates, on days −14 and −7. Controls were injected with HBSS. Hepatic metastases were established on day 0 through intrasplenic injections of 1 × 105MCA-106 tumor cells. Animals were sacrificed on day 21 and their livers were excised to assess tumor burden. Splenocytes from DC-treated groups were cytotoxic against MCA-106 cells, but not against the L929 and CT26 (syngeneic fibroblast and colon tumor, respectively) cell lines. All control mice developed grossly evident hepatic metastases, while 62 and 44% of the mice receiving MCA-106 cell lysate-pulsed DC and unpulsed DC vaccines, respectively, were completely free of tumor. Mean hepatic mass for the controls, including tumor, was almost double that for treated animals. Antibody depletion of either CD4+or CD8+lymphocytes abrogated the protective effect of the vaccine. This study demonstrates that immunization with DC confers cellular immunity, with both CD4+and CD8+T-cells playing a significant role, and impedes the subsequent establishment and growth of hepatic metastases in mice. The antitumor capabilities of DC justify their use in immunotherapeutic vaccines against human cancers.

Possible effect of pneumoperitoneum on the spreading of colon cancer tumor cells.

By using a murine hepatic metastatic model, we tried to investigate the possible influence of gas insufflation in colon cancer cells spreading from the portal system to the liver. After transducing the human placental ALP gene into murine colon cancer cell line CT26, we successfully selected a clone of CT26/DAP that would yield a specific color following histochemical staining. Fifty mice were assigned into two groups, receiving either an intrasplenic injection of 10(6) CT26/DAP cells alone or the cells followed by intra-abdominal helium insufflation with the pressure of 15 cm H2O for ten minutes. Five mice in each group were used to observe their survival and the other mice were killed at four different time periods: 10 minutes, 24 hours, 48 hours, and 72 hours following cell injection. The livers and spleens were removed for histochemical staining. By counting the numbers of specific dark reddish spots of CT26/DAP cells, we could estimate the number of tumor cells on the hepatic surface. At the very beginning following tumor cell injection, we found a significantly greater number of tumor cells on the hepatic surface in mice with gas insufflation (6354 +/- 1072 vs. 2133 +/- 223, respectively; P = 0.012). But the difference of these two groups became smaller and smaller as time went by. The number of tumor cells on the hepatic surface would reach the lowest level at postoperative 48 hours, and the tumor foci then began to grow both in size and number. The above patterns of dynamic change in tumor cell distribution were similar in mice both with and without gas insufflation. Average survival was slightly shorter in mice with gas insufflation, but the difference was not statistically significant. Pneumoperitoneum caused by gas insufflation may increase tumor cell spread from the portal system to the liver at the very beginning stage; however, there was no significant difference in long-term survival between mice with and without gas insufflation in this murine animal model.

Recombinant vaccinia interleukin-2-infected tumor cell vaccines in immunotherapy of murine colon adenocarcinoma.

A recombinant vaccinia virus (vCF13) containing and expressing the gene for human interleukin (IL)-2(vCF13) was compared to a recombinant vaccinia transfection control strain containing the LacZ gene at the same insertion site (vTFCLZ-1) for their ability to augment the immunogenicity of murine colon adenocarcinoma cell lines CT26 and CA51 in Balb/c mice. Both recombinant vaccinia strains abolished tumorigenicity of 10(5) CT26 or CA51 tumor cells. vCF13-infected tumor cells that secreted human IL-2 as measured by both CTLL-2 and enzyme-linked immunosorbent assays induced delay in tumorigenesis when administered in two weekly subcutaneous injections 1 week prior to challenge with 10(5) uninfected tumor cells. Although three of five vCF13-CT26 immunized mice developed tumors by day 14 after challenge, intralesional injection of these tumors with vCF13 induced rapid regression, whereas all five tumors that developed in vTFCLZ-1 immunized mice showed no response to intralesional vTFCLZ-1. These preliminary results provide support for the potential utility of recombinant vaccinia/IL-2 in tumor immunotherapy.

The effect of nonparabolic energy bands on tunneling through thin insulating films

Targeted clearance of colorectal cancer stem cells (CCSCs) has become a novel strategy for tumor immunotherapy. Molecule mucin1 (MUC1) is one of targetable cell surface antigens in CCSCs. However, the critical role of MUC1 in anti-tumor effects of CCSC vaccine remains unclear. In the present study, we showed that MUC1 may be required for CCSC vaccine to exert tumor immunity. CD133+CCSCs were isolated from CT26 cell line using a magnetic-activated cell sorting system, and MUC1 shRNA or recombinant plasmid was further used to decrease or increase the expression of MUC1 in CD133+CCSCs. Mice were subcutaneously immunized with the CCSC lysates, MUC1 knockin CCSCs, and MUC1 knockdown CCSCs respectively, followed by a challenge with CT26 cells. We found that CCSC vaccine significantly reduced the tumor growth via a target killing of CCSCs as evidenced by a decrease of CD133+ cells and ALDH+ cells in tumors. Moreover, CCSC vaccine markedly increased the cytotoxicity of NK cells and the splenocytes, and promoted the release of IFN-γ, Perforin, and Granzyme B, and also reduced the TGF-β1 expression. Additionally, CCSC vaccination enhanced the antibody production and decreased the myeloid derived suppressor cells and Treg subsets. More importantly, MUC1 knockdown partly impaired the anti-tumor efficacy of CCSC vaccine, whereas MUC1 overexpression dramatically enhanced the CCSC vaccine immunity. Overall, these results reveal a novel role and molecular mechanisms of MUC1 in CCSC vaccine against colorectal cancer.