Effects Of Lentivirus-mediated HIF-1 α Knock-down On Hypoxia-related Drug And Radiation Resistance And Their Dependence On P53 Status In Fibrosarcoma Cells

INTRODUCTIONHypoxia, a reduction in the normal level of tissue oxygen tension, inculding both acute and chronic hypoxia, is the most important and prevailing character of microenviroment in solid tumors, which is partly the results of unlimited growth of tumor cells, insufficient blood supply and angiodysplasia. A large pool of evidence have shown that hypoxia is closely associated with tumor resistance to radiation and drug as well as poor prognosis. As the most important transcription factor, hypoxia inducible factor-1 (HIF-1) plays important roles in many gene expression which allow the cancer cells to survive and even to proliferate under hypoxia, such as genes associated with neoangiogenesis, glycolysis and antiapoptosis, which make it one molecular target of great interests. Given the close crosstalk between microenvironment and tumor genes, especially the interaction between HIF-1 and tumor p53, targeted therapy towards HIF-1 is hypothesized to depend partly on the functional p53 staus. Since HIF-1αwas seen in 70% tumor primary lesions and metastasis, and p53 mutation was seen in more than 50% neoplasms, the interaction of HIF-1 with either wild type or mutant type p53 would be common in tumors and be also of great prognostic value.HIF-1 is a heterodimer HLH—PAS protein which consists of HIF-1αand HIF-1βsubunits.HIF-1β(ARNT) is constitutively expressed in some cells while HIF-1αis precisely regulated by cellular oxygen levels. Under hypoxic conditions, HIF-1αis induced, dimerizes with aβsubunit, translocates to the nucleus and initiates gene transcription of the targeting genes. In the presence of oxygen, prolyl hydroxylase (PH) which contains an iron moiety modifies the HIF-1 post-translationally to allow it combine with VHL complex, then HIF-1 undergoes ubiquitination and proteasome degradation in several minutes.Although HIF-1αis good indicative of poor outcomes in clinical data, the conclusion is still inconsistent on how and what roles HIF-1αplays in hypoxia related radiation and drug resistance. Different oxygen concentration used in experiments and cell lines might contribute to the inconsistency. HIF-1 knockdown-mediated radiation or drug sensitization was tested under normoxia in some studies with negative results, where HIF-1 was expected to play minor roles in the presence of oxygen and neither the cells with normal HIF-1 status nor cells with HIF-1 suppression would become the major part of therapy barrier. As far as we know, the effect of interaction of HIF-1 and p53 on radiation sensitivity was only reported in one study in PC-3 cells. Instead of radiosensitization, HIF-1 inhibition resulted in greater radiation resistance. Moreover, HIF-1 expression together with p53 mutation was indicative of the worst prognosis in gastic cancer patients in another study, which suggested great therapeutic importance of HIF-1 and p53 interaction. All the results warrant further reserch to verify them.Now, most studies on crosstalk between HIF-1 and p53 focused on the direct interaction between proteins, regluation on cell cycle, apoptosis and downstream genes expression. p53 mutation was associated with malignant phenotype, while HIF-1 could enhance such effects. At least part of proapoptotic action of HIF-1 was realized by inhibition of p53 degradation. On the other hand, wild type p53 could inhibit the transcription activities of HIF-1 and expression of downstream gene expression. However, few data were available about effects of such interaction on hypoxia related radiation and drug resistance.So, this study was aimed to explore the effects of HIF-1αknock-down on hypoxia-related drug and radiation resistance and their dependence on p53 status in fibrosarcoma cells using lentivirus packed shRNA. The underlying molecular mechanisms were also studied.. Part I Lentivirus-mediated HIF-1αknock-down on hypoxia-related drug and radiation resistance and their dependence on p53 status in fibrosarcoma cells1. Materials and methods1.1 Cell lines: HT1080 (p53 wild type), HT1080-6TG (p53 mutant type).1.2 HIF-1αRNAi lentiviral vectors were constructed with BLOCK-iT Lentiviral RNAi Expression System.1.3 RNAi lentiviral vectors were packaged by co-transfecting 293FT with 4 plasmids. The functional titre was determined with transduction assay.1.4 HIF-1αRNAi gene specific silenced HT1080 and HT1080-6TG were constructed by virus transduction and blasticidin screening.1.5 HIF-1αmRNA expression was determined by real-time PCR.1.6 HIF-1αprotein expression was determined by Western Blotting.1.7 Hypoxic treatment: Adjust oxygen concentration to be 0.5% using tri-gas incubator.1.8 Cell proliferation assay: Typhan blue exclusion cell number counting.2 Results2.1 HIF-1αRNAi lentival vector (lenti6-HIF1α) was successfully constructed. Thetransduction titre in HT1080 and HT1080-6TG was 7.43×10⁵TU/ml and 6.27×10⁵TU/ml respectively.2.2 HIF-1 a gene specific silenced HT1080 and HT1080-6TG cells were established and namedHT1080/ HIF-1α(-)and HT1080-6TG/ HIF-1α(-).2.3 Permenent transduction with HIF-1a RNAi lentiviral vector resulted in sequence specificsilencing with 86.7% and 84.4% decreases of HIF-1αmRNA under normoxia in HT1080 and HT1080-6TG respectively, while 91.6% and 97.3% uncer hypoxia.2.4 No significant difference among Ienti6-HIF1αtransducted cells and their parent cells under normoxia. However, HIF-1αprotein expression was decreased by 84.7% and 96.2% in HT1080/HIF1α(-) and HT1080-6TG/HIF1α(-) under hypoxia. 2.5 The doubling time among HT1080,HT1080-6TG, HT1080/HIF1α(-) andHT1080-6TG/HIF1α(-) was 25.96hrs, 26.35h, 26.34h and 24.15h respectively under normoxia. However, cell growth arrest was apparent under hypoxia in HT1080,HT1080-6TG, and HT1080/HIF1α(-), while HT1080-6TG/HIF1α(-) continued to grow at a slower rate.3 Conclusions3.1 The construction and production of lentiviral vectors was successful.3.2 The selected cells demonstrated significant disruption of HIF-1αexpression at both mRNA and protein levels.3.3 HIF-1αknock-down exerted no significant influence on cell growth under normoxia.3.4 The coexsistence of HIF-1 knock-down and mutation of p53 resulted in growth arrest resistance under hypoxia.Part II Effect of HIF-1αknock-down on hypoxia-related radiation and drug resistance and their dependence on p53 status1. Materials and methods1.1 Drug sensitivities to cisplatin and etoposide were assayed by MTT. IC₅₀ comparison between hypoxia and normoxia was used to calculate OER (oxygen enhancement ratio). The cells were preconditioned under hypoxia for 4 h and then cocultured with drugs for further 24h.1.2 Radiation sensitivity: Colony forming assay was used for evaluation of celluar sensitivity to radiation.Linear-quadratic model was used to radiation dose survival curve establishment. The cells was cultured under hypoxia for 24 hrs and then radiated in 10 mins after reoxygenation.1.3 Hypoxic treatment: Adjust oxygen concentration to 0.5% and 1.0% using tri-gas incubator.2 Results2.1 Sensitivity of both HT1080 and HT1080-6TG to etoposide and cisplatin was decreased under hypoxia. 2.2 Hypoxia-related drug resistance was oxygen concentration dependent. OER to cisplatin under 0.5% and 1% oxygen was 2.47±0.26 and 1.87±0.13 for HT1080, P<0.05. For HT1080-6TG, OER was 1.72±0.02 and 1.18±0.11, P<0.05.2.3 Hypoxia-related cisplatin resistance was dependent.on p53 status. OER under 0.5% oxygen for HT1080 and HT1080-6TG was 2.47±0.26 and 1.72±0.02, P<0.05.2.4 Hypoxia-related etoposide resistance was independent.of p53 status. OER under 0.5% oxygen for HT1080 and HT1080-6TG was 2.09±0.51 and 1.71±0.42,P>0.05.2.5 HIF-1αknock-down could reverse hypoxia-related cisplatin resistance in HT1080 cells, while had little effect on etoposide sensitivity. OER for cisplatin in HT1080/HIF1α(-) was 1.47±0.33, vs. 2.47±0.26 (HT1080), P<0.05.2.6 HIF-la knock-down failed to reverse hypoxia-related cisplatin and etopside resistance in HT1080-6TG cells.2.7 Posthypoxia radiation resistance could be seen in both HT1080 and HT1080-6TG with OER to be 1.24 and 1.23 respectively.2.8 HIF-1αknock-down could reverse hypoxia-related radiation resistance in HT1080 cells with OER to be 0.75, while had little effect on HT1080-6TG with OER to be 1.16.3 Conclusions3.1 Hypoxia-induced drug resistace was dependent on oxygen concentration, drugs, and p53 function.3.2 Reversal effect of hypoxia-induced drug and radiation resistance by HIF1αknock-down was partly dependent on normal p53 function.Part III Molecular mechanisms of effects of HIF-1αknock-down on reversal hypoxia-induced drug and radiation resistance 1 Materials and methods1.1 Cell cycle analysis: Flowcytometry was used for analysis of DNA contents with PI staining and Modtif software was used for analysis of cell cycle.1.2 Apoptosis analysis: Flowcytometry was used for apoptosis analysis with Annexin-V/ PI and Caspase 3 staining.1.3 Western blotting was used for HIF-1αand p53 signaling downstream protein expression.2 Results2.1 Hypoxia (0.5% oxygen for 24h) induced cell cycle arrest in both HT1080 and HT1080-6TG with G1 phase increasing and S phase decreasing. Such effects became inapparent when HIF-1αwas knocked down, especially for HT1080-6TG/HIF1α(-).2.2 No great difference was seen in apoptotic index under hypoxia (0.5% oxygen for 24hrs) among HT1080, HT1080-6TG and their corresponding cells with HIF-1αknocked down. However, apoptosis was dramatically enhanced with the addition of cisplatin(20μM) for 24h. Hypoxia protected the cells from cisplatin-induced apoptosis in both HT1080 and HT1080-6TG cells, whose protective effects was lost when HIF-1αwas knocked-down in HT1080/ HIF1α(-), but still worked in HT1080-6TG/HIF1α(-).2.3 Under normoxia, only bcl-2 showed redution in basal expression levels in cells with HIF-1 knocked-down. Under hypoxia(0.5% oxygen for 24h), no significant changes was seen in bcl-2, while phophos-p53 and p21 expression increased in HT1080. Hypoxia increased bax expression in HT1080-6TG, while HIF-1αknock-down attenuated this effect in HT1080-6TG/HIF1α(-). By contrast, Bax increasing was more apparent under hypoxia in HT1080/HIF1α(-) than its parent cells. In comparison with their parent cells, bid expression was enhanced when HIF-1αwas silenced under hypoxia, especially in HT1080/HIF1α(-).3 Conclusions3.1 Hypoxia-induced cell cycle arrest was determined by both HIF-1 and tumor suppressor p53. Such effects were attenuated in the presence of both HIF-1 knock-down and p53 mutation. 3.2 Hypoxia protected cells from drug-induced apoptosis, whose protective effects were lost in wild type p53 HT1080 cells when HIF-1 was silenced.3.3 Hypoxia-induced drug and radiation resistance might be related to expression changes of many downstream genes in both HIF-1αand p53 signalling pathway.