The Influence Of Antisense Oligonucleotides Targeting HIF-1α On Hep-2 Cells And Enhance Radiotherapeutic Activity Against Hep-2 Cells In Vitro

Rapid growth in hypoxia environment is a common characteristic of the tumor in human beings and animals. The partial pressure of oxygen in most of the entity tumors is lower than that of normal tissue. Hypoxia is one of the necessary environmental conditions during the process of tumor growth. Experimental and clinical data have demonstrated that hypoxia is related to the tumor malignancy, and can affect gene stability, tumor apoptosis, angiogenesis and metastasis. Hypoxia is an independent factor leading to poor prognosis of most malignant tumors, also an important factor that makes tumors resist and tolerate therapies including radiotherapy and chemotherapy.Hypoxia induced factor 1α(HIF-1α) is a kind of intermediate hypoxic adaptable transcription factor which lies in mammals and human cells. Research indicates that it widely lies in many kinds of human tumor cells. It is a key protein that directly reacts to the oxygen deficit. With the decrease of oxygen concentration in surrounding tissues, the expression of HIF-1αmay increase. HIF-1αplays important roles in maintaining the energy metabolism for tumor multiplication, invasion and metastasis. The protein over-expression of HIF-1αexists in many kinds of malignant tumors, which closely related to the poor prognosis and resistance and tolerance to radiotherapy or chemotherapy. The technique of antisense oligodeoxynucleotide(AS-ODN) is one of antisense gene therapy methods most commonly used. Basing on the principle of base complementation, the double strands of heterozygotic DNA-RNA is formed through binding specifically the antisense DNA fragments and definite base sequence of the aimed mRNA. It can induce RNA enzyme to catalyze mRNA hydrolysis and then block up mRNA's shearing, transporting and protein interpretation. It was reported that HIF-1αAS-ODN can down-regulate the micro-vessel density of tumor and completely remove the tumor less than 0.1cm in diameter. With the increasing acknowledgement of the influence and control factor about HIF-1α, a series of methods which can down-regulate the level of expression of HIF-1α, are likely to become a new method of treatment for cancer.The head and neck squamous carcinoma, as one of the most common malignant tumors, strongly resists to chem- and radio-therapy. Its treatment has been a tough problem to solve. Up to now in vitro study on the relationship between HIF-1αand radio-sensitivity of squamous carcinoma of head and neck has not been reported. Therefore investigation of the role of HIF-1αon genesis, development and metastasis of squamous carcinoma of head and neck in hypoxia situation, can help us to find a new way to treat cancers by interfering the effect of HIF-1α.This study includes three parts:PART one: Impact of hypoxia on Hep-2 cell proliferation, apoptosis, the expression of HIF-1αand its correlative genes p53 and VEGF.ObjectiveTo investigate the influence of hypoxia on the expression of HIF-1αprotein of Hep-2 cells of human laryngocarcinoma, and to further explore the correlationship between HIF-1αand changes of hypoxia on Hep-2 cell proliferation, apoptosis, angiogenesis.Methods1. Hep-2 cells of human laryngocarcinoma were incubated in vitro under the normoxia(37℃, 5%CO2, 20%O2) and hypoxia condition (37℃,5%CO2,2%O2). Survival fraction(SF) of Hep-2 cells was determined by MTT. Flow cytometry(FCM) was used to measure the distribution of cell cycles and to detect cell apoptosis.2. The expression of HIF-1αmRNA in Hep-2 cells was detected at different time points by reverse transcription-polymerase chain reaction (RT-PCR).3. Immunohistochemistry was used to detect the expression of HIF-1αand p53 protein at different time intervals.4. The expression of HIF-1αand VEGF protein was detected at different times by FCM.Results1. The results by MTT showed that, after exposure to the hypoxia condition for 6h, 12h, 24h, 36h, SF of Hep-2 cells were higher than that in normoxia groups. There was a significant difference in SF between groups of normoxia and hypoxia (P<0.05). But there was no difference in statistic in SF between group of hypoxia for 48h and group of normoxia for 48h(P>0.05).2. By FCM, the results of the distribution of cell cycles in Hep-2 cells showed that, after incubation under the hypoxia condition for 6h, 12h, 24h, 36h, proliferation index (PI) and S-phase cell fraction (SPF) increased gradually, and the ratios of G0/G1 decreased gradually. There was a significant difference between groups of normoxia and hypoxia (P<0.05). But under hypoxia for 48h, PI and SPF decreased remarkably.3. After incubated under the hypoxia condition for 6h, 12h, 24h, 36h, apoptosis rate of Hep-2 cells was remarkably lower than under the normoxia condition at each time point(P<0.01). But there was no difference of apoptosis in statistic between group of hypoxia for 48h and group of normoxia for 48h (P>0.05).4. The results by RT-PCR showed that, there was no significant difference for expression of HIF-1αmRNA in between different groups (P>0.05).5. The results of immunohistochemistry experiment showed that, HIF-1αprotein expressed in cytoplasm of Hep-2 cells cultured in hypoxia condition, the longer Hep-2 cells were cultured in hypoxia (within 36h), the higher HIF-1αprotein expressed. Partial expression of HIF-1αin nucleus could also be seen in some cells. The positive expression of HIF-1αprotein in hypoxia group was significantly higher than that in normoxia group (P<0.01). P53 protein expressed in nucleus of Hep-2 cells. The expression of p53 protein increased following the prolongation of hypoxia time (within 36h). There was significant difference for expression of p53 protein between hypoxia group and normoxia group (P<0.01). Through Pearson correlation analysis, there was a positive correlation between expression of HIF-1αand p53(r=0.655, P<0.05).6. The expression of HIF-1αand VEGF protein detected by FCM increased following the prolongation of hypoxia time (within 36h), and they both decreased in 48h of hypoxia. Through Pearson correlation analysis, there was a positive correlation between expression of HIF-1αand VEGF(r=0.815, P<0. 05).Conclusions1. The expression of HIF-1αprotein in Hep-2 cells of human laryngocarcinoma increases under the hypoxia condition. It increases gradually following the prolongation of hypoxia. Under hypoxia condition, the expression of HIF-1αmRNA in Hep-2 cells is not up-regulated. That is, under hypoxia condition the increase of its expression happens in protein level.2. Within a period of hypoxia time, apoptosis of Hep-2 cells is inhibited, and proliferation is enhanced. HIF-1αmight play important roles in G0/G1 cell cycle defect and S cell cycle arrest induced by hypoxia.3. The expression of HIF-1α, p53 and VEGF protein gradually increase following the prolongation of hypoxia time. There are positive correlations between expression of HIF-1αand p53, VEGF. HIF-1αmight play important roles in resistance of cells to apoptosis and in tumor angiogenesis.PART two: The influence of radiation on the expression of HIF-1αprotein and the sensitivity of Hep-2 cells to radiation (γ-rad) under hypoxiaObjectives.To investigate the effects of radiation on the expression of HIF-1αprotein of Hep-2 cells of human laryngocarcinoma under hypoxia, and to further explore the impact of over-expression of HIF-1αprotein in hypoxia on the sensitivity of Hep-2 cells to radiation (γ-rad).Methods1. Hep-2 cells incubated under the normoxia and hypoxia conditions receivedγ-rad at a graded series of radiation doses(2Gy, 4Gy, 8Gy). By crystal violet and FCM, we examed respectively the cell proliferation, apoptosis and expression of HIF-1αprotein in Hep-2 cells incubated under different conditions and different doses ofγ-rad.2. Hep-2 cells incubated under the normoxia and hypoxia conditions received 4Gyγ-rad. After 24h, cell cycle of Hep-2 cells was detected by FCM.3. Hep-2 cells incubated under the normoxia and hypoxia conditions receivedγ-rad at a graded series of radiation dosage(2Gy, 4Gy, 8Gy). After 24h, the expression of HIF-1αmRNA in Hep-2 cells was detected by reverse transcription-polymerase chain reaction (RT-PCR). Results1. In radiation groups with both normoxia and hypoxia, the cell inhibition rate (IR) and apoptosis rate(AR) increased gradually following the raise of radiation doses(0, 2, 4, 8Gy), there were significant difference among the groups with different doses ofγ-rad (P<0.05). With the same dosage ofγ-rad, IR and AR in group of hypoxia was lower than that in group of normoxia, the difference was statistically significant(P<0.01). After 4Gy ofγ-rad, IR and AR in group of hypoxia were lower than those in group of normoxia, the difference was statistically significant(P<0.01).2. In radiation groups with both normoxia and hypoxia, the ratios of G0/G1 increased, and PI decreased. There was a significant difference between groups of radiation and groups of no-radiation(P<0.05). But influence of hypoxia on cell cycle was more remarkable than that of radiation.3. The results by RT-PCR and FCM showed that, there was no significant difference for expression of HIF-1αmRNA and protein between groups of radiation and groups of no-radiation (P>0.05).4. Through Pearson correlation analysis, there was a positive correlation between the expression of HIF-1αand the decrease of inhibition rate and apoptosis rate which was reduced by radiation in hypoxia(r=0.971, 0.979, P<0.01). There was a negative correlation between the decrease of inhibition rate and IAR which was reduced by radiation in hypoxia and the ratios of G0/G1 cell cycle(r=-0.753, -0.815, P<0.01).Conclusions1. Effects of hypoxia on cell cycle are more remarkable than that of radiation. So the change of cell cycle in hypoxia might be one of the major factors which induced Hep-2 cells resisting to radiation.2. Hypoxia can decrease the sensitivity of Hep-2 cells to radiation(γ-rad), by decreasing apoptosis and increasing resistance of cell to radiation through up-regulating the related gene expression as HIF-1α.PART three: The effect of HIF-1αAS-ODN on the expression of HIF-1αand the sensitivity of Hep-2 cells to radiationObjectivesTo explore effects and the mechanism by which antisense oligonucleotides targeting HIF-1αinduce apoptosis, and to search a feasible way to enhance radio-therapeutic activity against Hep-2 Cells.Methods1. HIF-1αAS-ODN was designed and synthesized, which was complementary with initiation codon region of HIF-1αmRNA. After embedded by cation lipofectamine, it was transfected into Hep-2 cells of human laryngocarcinoma, Then the transfected cells were incubated under hypoxia condition(37℃, 5%CO2, 2%O2). The Hep-2 cells rate of proliferation inhibition (IR), rate of apoptosis (AR), the distribution cell cycle and the expression of HIF-1αmRNA and protein were detected by MTT, FCM, RT-PCR and western-blot.2. After transfection with 200nmol/L HIF-1αAS-ODN, immunohistochemistry was used to detect the expression of HIF-1αand p53 protein at 24h. The expression of HIF-1αand VEGF protein was evaluated at different times by FCM.3. After transfection with 200nmol/L HIF-1αAS-ODN, Hep-2 cells were treated with 4Gyγ-rad, the Hep-2 cell proliferation inhibiting rate(IR) and apoptosis rate(AR) were detected by MTT and FCM at different times(12, 24, 36, 48h).Results1. After transfection with different concentrations of HIF-1αAS-ODN, the shape of Hep-2 cells transformed to different degree, including decreasing in cell volume, abnormal-shape, cells shrinkage and rough cytoplasm. At the same time, the Hep-2 cells in the control group displayed normal shape and adherence.2. IR, AR and the ratios of G0/G1 increased and PI decreased with increasing of the concentration of HIF-1αAS-ODN transfection. There were significant difference between AS-ODN groups and controls(S-ODN group, LP2000 control group, blank control group) (P<0.01). While HIF-1αS-ODN of 400nmol/L and 600nmol/L also inhibited Hep-2 cells proliferation.After 200nmol/L of HIF-1αAS-ODN transfection for 12h and 24h, IR and AR in transfection group were remarkable higher than those in control group, the difference was statistically significant(P<0.01). After 36h, IR, AR and the ratios of G0/G1 in transfection group decreased.3. With increasing of the concentration of HIF-1αAS-ODN transfection, there was a decreasing tendency for the expression of HIF-1αmRNA and protein detected by RT-PCR and western-blot. There was significant difference of expression of HIF-1αbetween AS-ODN group and control group(S-ODN group, LIP2000 control group, blank control group), but there was no significant difference of the expression of HIF-1αin the 3 groups(P>0.05). After 200nmol/L of HIF-1αAS-ODN transfection, the most remarkable decreased after transfection for 24h.4. With 200nmol/L of HIF-1αAS-ODN transfection for 24h, the expression of HIF-1αand p53 protein in AS-ODN group detected by FCM were lower than that in control groups (S-ODN group, LP2000 control group, blank control group) (P<0.01). The results detected by SP showed that, after transfected with HIF-1αAS-ODN, the expression of HIF-1αand VEGF protein in Hep-2 cells was down-regulated, its expression in Hep-2 cells of HIF-1α AS-ODN group were significantly lower in statistic than that in other three groups (S-ODN group, LIP2000 control group, blank control group) (P<0.01). Based on the correlation analysis, there was positive correlation between the expression HIF-1αand p53 and between HIF-1αand VEGF.5. With 200nmol/L of HIF-1αAS-ODN transfection followed by 4Gyγ- rad at different time after transfection, IR and AR in AS-ODN plus radiation group were remarkably higher than that in any other groups(AS-ODN group, radiation group, S-ODN group, blank control group). The most remarkable increased after transfection for 24h.Conclusions1. The expression of HIF-1αprotein and mRNA in human Hep-2 cells can be inhibited and blocked effectively by HIF-1αAS-ODN in a dose dependent manner.2. Study of antisense oligodeoxynucleotides targeting HIF-1αshowed that HIF-1αAS-ODN transfection played an important role in increasing ratio of G0/G1, cell cycle arrest, augmenting the effect of rad-induced Hep-2 cell apoptosis and decreasing SPF. So HIF-1αAS-ODN enhances the sensitivity of Hep-2 cells to radiation.The expression of HIF-1α, P53 and VEGF protein increased under hypoxia condition. Proliferation and resistance apoptosis of Hep-2 cells was enhanced. The changes of Hep-2 cells in hypoxia led to decrease of radio-sensitivity. By HIF-1αAS-ODN transfection, radio-sensitivity of Hep-2 cells increased. This study has been not reported. The data play an important role in further treatment of laryngocarcinoma.