Effects Of Antisense Oligonucleotide Targeting Osteopontin On Invasion And Metastasis Of Colon Carcinoma Cell Lines HT-29 In Vitro Under Moderate Hypoxia

Liver metastasis is the main cause that influences the five-year survival rate after radical excision of primary colorectal carcinoma. If the cases high risk to liver metastasis could be detected by a simple biological indicator, the measures are taken to treat it, which is siginificant to improve the survival of colorectal carcinoma.The local infiltration and distant metastasis is the most important feature of malignant tumor. The microenvironment in stroma plays a regulative role in the malignant progression of solid tumor. Hypoxia is the basic characteristic of the physical microenvironment in solid tumor. Moderate hypoxia might be the initiating agent that caused the instability of heredity and promote the malignant transformation. OPN is a highly phosphorylated and glycosylated protein which has been shown to mediate the invasiveness of tumor. OPN was up-regulated in a time-dependent manner induced by hypoxia. Being short of hypoxia response element (HRE), osteopontin is under no direct control of HIF-1α. With regard to its multiple functions, we assume that osteopontin is the other regulative factor besides HIF-1αunder moderate hypoxia.As a common solid tumor of digestive system, malignant progression of colorectal carcinoma carries out in hypoxic condition too. At present, the biological behavior of colon carcinoma cells in hypoxic condition has been poorly known. Furthermore, there are few reports that involve exploreing the correlation between osteopontin and invasiveness through simulateing hypoxic microenviroment in vivo.We focused on morphologic character at invasive front of colorectal carcinoma in combination with the expression of osteopontin within it so as to study relationship between these factors and liver metastasis. Moreover, this study was taken colon carcinoma cell lines HT-29 as an object, hypoxic conditions in vitro were produced according to PO2 of tumor in vivo. We observed the biological behavior of colon carcinoma cells in hypoxic condition. Correlation between osteopontin and invasiveness induced by moderate hypoxia was explored. So, we tried to investigate the correlation between moderate hypoxia and maliglant transformation.Part 1 Expression of Osteopontin at the Invasive Front of Primary Colorectal Carcinoma with Liver MetastasisObjective: To explore the distribution of tumor budding and the expression of osteopontin in primary colorectal cancer and tumor budding in order to evaluate whether that could be used as correlation factor for liver metastasis after surgery to remove colorectal carcinoma.Methods: Cases diagnosed as Dukes stage C or D were divided into three groups, namely, group A with synchronous liver metastasis, group B with heterochronous liver metastasis, and group C with no liver metastasis. After H&E staining, attention was focused on observing the distribution characteristics of tumor budding at the invasive front of colorectal carcinoma and recording the numbers accurately. The expression of osteopontin and NF-κB in primary lesions and tumor buds was examined using immunohistochemistry.Result: Tumor budding was defined as depolarized and dedifferentiated cancer cells with isolated and irregular trabecular form ahead of the invasive front of the lesion. Both the degree of tumor budding and the strong positive expression of osteopontin in primary colorectal cancer showed a significant difference only between group A and group B (p<0.01).The positive expression of osteopontin in tumor buds showed a significant difference between groups A and C and between groups B and C (p<0.01).As for the expression of NF-κB, there is no significant difference among 3 groups.Conclusion: Both tumor budding degree (BD)(+) and the strong positive expression of osteopontin in primary colorectal cancer indicate increased risk of liver metastasis following colorectal carcinoma. If the distribution characteristics of tumor budding are assessed in combination with the expression of osteopontin within the tumor buds, the predictive accuracy is increased and can be used as correlation factor for liver metastasis after surgery to remove colorectal carcinoma.Part 2 Establishment and Identification of Hpoxic Model as well as the Influence of Hypoxia on Growth of Colon Carcinoma Cell Lines HT-29Objective: To establish and assess a simple and stable hypoxic model in vitro according to PO₂ of tumor in vivo. To investigate the effect of various PO₂ of culture on growth of cells and the distribution of cell cycle in order to find out a moderate hypoxia fit to proliferation.Method: After pouring compound gas at various oxygen concentrations into self-made hypoxia monitoring unit, PO₂ of culture was detected instantly by resolve oxygen apparatus. The HIF-1αmRNA levels of HT-29 cells were examined by RT-PCR so as to validate hypoxic condition. The morphologic changes of HT-29 cells under hypoxia were observed by inverted phase contrast microscope. The proliferation of HT-29 cells was detected using MTT color metric assay in order to draw a growth curve. The distribution of cell cycle and apoptosis of HT-29 cells were assessed by flow cytometry. Result: After pouring into compound gas at oxygen concentration of 7.8%, 4.8%, 2.4% and 1.2% for 20min, the PO₂ of culture were constantly 30mmHg, 20mmHg, 10mmHg and 5mmHg respectively. The expression of HIF-1αmRNA under hypoxia was significantly increased as compared with the control, among which the highest levels of HIF-1αmRNA occurred in PO₂20mmHg group. After 24hours hypoxia, the morphous of HT-29 cells in PO₂30mmHg, PO₂20mmHg and PO₂10mmHg group were similar to the control, while HT-29 cells in PO₂5mmHg group appeared morphologic changes impaired by severe hypoxia. The absorbance values of HT-29 cells in PO₂20mmHg group were higher than that of control group with statistic significance, while those in PO₂30mmHg and PO₂10mmHg groups increased too, but without statistic significance as compared with the control. The proliferation of cell in PO₂5mmHg was the lowest among the groups with the significant difference. The ratio of G₂/M in PO₂20mmHg group was similar to the control, however, the ratio of S phase were increased obviously with statistic significance. As for PO₂5mmHg group, the apoptosis of HT-29 cells was significantly increased. Severe hypoxia could distinctly induce cell cycle arrest in G₀/G₁ phase, Ratio of S phase was decreased markedly with statistic significance as compared with the control. The distribution of cell cycle in PO₂30mmHg and PO₂10mmHg groups was different from that of the control but without statistic significance. Conclusion: Hypoxic model in vitro was established successfully. Under moderate hypoxia, the hypoxia response gene was up-regulated oweing to stress effect of hypoxia. The process of apoptosis was inhibited, simultaneously, proliferation of cells was induced to speed up, through which tumor cells may survive in hypoxic condition. On the other hand, severe hypoxia could induce cell cycle arrest in G₀/G₁ phase. It could also impair cells through promoteing apoptosis and inhibitting proliferation. The cells in PO₂20mmHg showed a strongest proliferation among the groups because the hypoxic level situated the point of balance between stress effect of hypoxia and damaged effect of that. The partial oxygen pressure at 20mmHg in vivo was situated at the verge of the tumor body, which suggested that the tumor cells at this site should be of stronger proliferation induced by moderate hypoxia. This result also established a fine base for further study to evaluate the invasiveness of HT-29 cells in this condition and explore its latent mechanism of maliglant transformation.Part 3 Moderate Hypoxia Up-regulates the Expression of Osteopontin and NF-κB and Augments Invasive Capacity in Colon Carcinoma Cell Lines HT-29Objective: To observe invasive capacity in colon carcinoma cell lines HT-29 under moderate hypoxia, and detect the expression of osteopontin, NF-κB/p65 protein in nucleus and activities of MMP-2/9 so as to explore its latent mechanism of maliglant transformation.Method: Hypoxic conditions were produced refering to the PO₂ at the verge of tumor in vivo (20mmHg). The heterotypic adhesiveness of HT-29 cells was detected by MTT assay. Reaction to a hypoxic environment was determined via invasion across a Matrigel-coated Transwell filter; Tube formation of human umbilical vein endothelial cells (HUVECs) were adopted to evaluate the abilitity of tumor angiogenesis in vitro; After 0, 6, 12, 24 or 48h of moderate hypoxia, MMP-2/9 activities secreted by HT-29 cells were assessed by gelatin zymography, and osteopontin mRNA and protein levels detected by RT-PCR and Western blot respectively. Similarly, NF-κB/p65 levels in nucleus were measured by Western blot after extraction of nucleic protein.Result: The adhesiveness of HT-29 cells gradually gained up with prolonging time. The cells treated with moderate hypoxia obviously had a higher adhesive rate than that of the control at different duration. The numbers of HT-29 cells migrated to the microporous membrane within 24 hours were: control group with 103.7±6.2; moderate hypoxia group with 185.6±8.4. The numbers of tubes formed by HUVECs were: control group with 12.4±2.8, moderate hypoxia group with 28.5±3.6. MMP-2/9 activities under moderate hypoxia at 6 h showed no significant change as compared with that under normoxia, then gradually up-regulated and reached the peak at 24h, they show no significant difference beween 24h and 48h after hypoxia. The expression trend of osteopontin mRNA, protein and NF-κB/p65 protein in nucleus was similar to that of MMP-2/9 under moderate hypoxia.Conclusion: Moderate hypoxia could induce maliglant transformation in HT-29 cells through enhancing heterotypic adhesiveness, invasive capacity and tumor angiogenesis and up-regulating MMP-2/9 activities. There was a general and synchronous up-regulation of osteopontin and NF-κB/p65 and MMP-2/9 in a time-dependent manner. OPN-NF-κB may be an important modulatory pathway besides HIF-1αunder hypoxic condition, which involves maliglant phenotype induced by moderate hypoxia.Part 4 Effects of Antisense Oligonucleotide Targeting Osteopontin on Invasion and Metastasis of Colon Carcinoma Cell Lines HT-29 in vitro under Moderate HypoxiaObjective: To study the effects of antisense oligonucleotide (ASODN) targeting osteopontin on the invasiveness of HT-29 cells under moderate hypoxia. To investigate correlation between osteopontin and malignant phenotype induced by moderate hypoxia.Methods: ASODN targeting osteopontin were selected refering to report by Adwan and synthesized with a phosphorothioate backbone. Mediateing by Lipofectamine, ASODN were transfected into HT-29 cells with high expression of osteopontin induced by moderate hypoxia. The specificity and dose-effect relationship between ASODN and osteopontin protein were examined by Western blot. Osteopontin mRNA levels of HT-29 cells treated with ASODN were detected by RT-PCR. The proliferation of HT-29 cells was assessed using MTT color metric assay. The heterotypic adhesiveness, migration, tumor angiogenesis, MMP-2/9 activities and NF-κB/p65 protein in nucleus were evaluated by methods similar to that described in detail in part three.Result: Compared with other controls, ASODN targeting osteopontin inhibited the expression of osteopontin protein in a dose-dependent manner. However, this tendency between levels of 10umol/L and of 20umol/L declined and showed no significant difference. ASODN targeting osteopontin could selectively and significantly down-regulate the levels of osteopontin protein and mRNA, with 35.4% and 31.5% of the controls respectively. The absorbance values in ASODN group were lower than that of the controls with a significant difference. The cellular inhibition rate went up to 41.6±1.2%. The cells treated with ASODN targeting osteopontin obviously had a lower adhesive rate than that of the controls at different duration. The numbers of HT-29 cells migrated to the microporous membrane and of tubes formed by HUVECs in ASODN group were only 52.6±3.8 and 12.4±2.8 respectively. ASODN targeting osteopontin could suppress significantly the MMP2/9 activities and NF-?B/p65 levels in nuclus of HT-29 cells exposed to moderate hypoxia. The above results showed a significant difference between ASODN group and other controls.Conclusion: ASODN targeting osteopontin could down-regulate the osteopontin mRNA and protein levels in HT-29 cells exposed to moderate hypoxia. The strong proliferation and heterotypic adhesiveness and angiogenesis and invasive capacity previously induced by moderate hypoxia could be inhibited by ASODN targeting osteopontin. It suggested that osteopontin played a key role in maliglant phenotype when HT-29 cells were exposed to moderate hypoxia. The levels of NF-κB/p65 in nucleus and MMP-2/9 activities were decreased when osteopontin were suppressed by ASODN. It also suggested that NF-κB and MMP-2/9 could involve maliglant phenotype induced by moderare hypoxia and could be regulatory downstream genes of osteopontin. In conclusion, OPN-NF-?B could be the other important regulatory pathway besides HIF-Iαunder moderate hypoxia. With regard to its multi-functions, the pathway could play a key role in the changes of biological behavior, especially in the regulation of malignant transformation when cancer cells were exposed to moderate hypoxia. Osteopontin might be an appropriate therapeutic target for the treatment of cancer utilizeing moderate hypoxic character of tumor in vivo.