Detection And Interference Of Low Perfusion And Hypoxia In Lung Cancer And Possible Molecular Mechanism

Part ONE Detection and interference of low perfusion and hypoxia in lung cancer.BackgroundAngiogenesis is defined as the process of developing new blood vessels and important to tumor formation and progress.Exiting research shows that the solid tumor cannot growth bigger than 2 mm in diameter without any support of vascular. Folkman J.et al found in transgenic animal model that there are angiogenesis accompanied with the progress from in situ to infiltrating stage.More and more clinical researches confirmed the relationship between tumor growth,metastasis, prognosis and angiogenesis.Recent years,anti-angiogenesis therapies attract more attention than ever.Many kinds of anti-angiogenesis substances are in their way. There are several ways to assay angiogenesis in tumor such as CT/MRI perfusion, SPECT.Most of them related to tumor of brain and liver while less of them for lung cancer.Current researches mainly focused on high perfusion area in tumor.Few of them focused on low perfusion.Low perfusion in lung cancer is of same importance as high perfusion.In microscopic facet,A.Low perfusion decrease the transportation of chemotherapy drug to solid tumor,which leads to uneven distribution of drug and low blood drug level in tumor.B.Low perfusion result in hypoxia,which leads to many changes in gene and protein levels that make tumor cell resistance to chemotherapy and radiotherapy.C.Tumor cells in hypoxia area are more liable to metastasis.In macroscopic facet,the growth and metastasis of tumor is the result of selection and adaption between the tumor and host.The tumor cells in low perfusion and hypoxia area can not get enough nutrition would necessarily select hypoxia resistance cells too survive and make part of them go far away to find new survival space.If we just use anti-angiogenesis drug,it is hard to block all the factors modulate angiogenesis.This may even accelerate the selection of more powerful tumor cell.You kill most tumor cells but got a few super-power cells for relapse in future.Hypoxic areas arise as a result of an imbalance between the supply and consumption of oxygen.Hypoxia-induced proteome and/or genome changes may promote tumor progression via mechanisms enabling cells to overcome nutritive deprivation,to escape from the hostile environment and to favor unrestricted growth. Sustained hypoxia may also lead to cellular changes resulting in a more clinically aggressive phenotype.The tissue hypoxia assay methods include positron emission tomography(PET),magnetic resonance imaging(19F MRI and BOLD MRI).PET is too expensive and need toxic probe,which limit its application.BOLD MRI is designed for brain function imageing.It is safe and convenient.The current anti-hypoxia therapy methods are mainly designed for radiotherapy.Few article report new way to improve chemotherapy resistance in hypoxia.Rhizomes of Paris polyphylla Smith vat.chinensis Hara has been used to treat cancer in China for many years.Its extractions such as polyphyllin D have been proved to have anti-tumor effect in vitro.To sum up,low perfusion and hypoxia in lung cancer are important to tumor growth,progressing and metastasis.Our hypothesis is angiogenesis and oxygen modulation other than simple anti-angiogenesis will work better to cure cancer.Paris polyphylla may have such effect.MRI perfusion and BOLD will be used as good way for lung cancer prognosis.Objective1.To establish a rabbit xenograft lung cancer model,assess effect of low perfusion and hypoxia on tumor prognosis.2.To observe the characteristic of tumor cell in low perfusion and hypoxia area.3.To observe the anti-tumor effect of Paris polyphylla,especially its effect on perfusion and hypoxia.Methods1.New Zealand rabbit were used in all experiments.VX2 lung cancer model was established in rabbits by tissue clot suspension injection.2.To verify the accuracy of MRI perfusion and BOLD result,3 tumor-bearing rabbits were used.OxyLite probes were inserted into 4 spots for every tumor guided by MRI image to measure the oxygen partial pressure at same time. 3.To test the relation between MRI perfusion/BOLD result and prognosis.10 lung cancer rabbit models were established as described.MRI scan was carried out on 10.20.30 and 45 days after injection.MRI perfusion/BOLD were done on 20th day.4.To observe the characteristic difference between tumor cells from low perfusion/hypoxia area and non-hypoxia area.MRI perfusion.BOLD and CT were carried out on a hind limb tumor bearing rabbit.Samples from low perfusion/hypoxia area and non-hypoxia area were taken by CT guided biopsy and made to A and B tumor cell suspension.4.1 The cell suspension A and B were subcultured in hypoxic and non-hypoxic condition for 48 hours.Real time RT-PCR,MTT and ELISA for apoptosis were carried out.4.2 20 rabbits were separate to two group(A and B) and given suspension A and B injection accordingly.MRI was carried out every ten days.CT guided biopsy were used to take sample for real time RT-PCR.5.To observe the effects of Paris polyphylla on VX2 tumor growth,especially on hypoxia and perfusion,hypoxic cell suspension was prepared as step4.A.the hypoxic cells were subcultured for 48h in both hypoxic and non-hypoxic condition with and without Paris polyphylla extraction added.Cell proliferation,apoptosis and gene expression were assayed as described above.B.50 rabbits were separated to five groups(group D:High dosage of Paris polyphylla.E:High dosage of Paris polyphylla plus 5-FU.F:5-FU.G:Low dosage of Paris polyphylla.H.control).The therapy effects were evaluated as described.Result:1.90%of rabbits undergone the suspension injection established lung cancer as desired.2.There is good correlation between results of MRI BOLD and OxyLite probe.3.High perfusion ratio is in positive correlation with tumor size and growth rate. Low perfusion ratio is in positive correlation with tumor metastasis and in negative correlation with life span.The area of high BOLD signal correlates to tumor metastasis and life span. 4.Under hypoxic condition,tumor cells from hypoxic area(group A) has higher proliferation rate than that from non-hypoxic area(group B).Real time RT-PCR shows that HIF1and VEGF expression in Group A were higher than group B.5.1) Paris polyphylla extraction has decreased the cell proliferation rate in normal oxygen level.This effect is more obvious in hypoxic condition.HIF1 and VEGF expression levels were down regulated in hypoxic condition by Paris polyphylla extraction.2) Paris polyphylla treated tumor bearing rabbit(group D) got small tumor size compared to control(group G),but not as much as in 5-FU treated rabbit (group F).Hypoxic area by MRI BOLD is less in Paris polyphylla treated groups(group D,E,G) compared with group F and H.Group E has the best outcome.Conclusion1.Tissue clot suspension injection is a reliable method to establish VX2 rabbit lung cancer model.2.Low perfusion and hypoxia area by MRI is useful for tumor prognosis.3.Tumor cells in Low perfusion and hypoxia area have special characteristics and should be key point for relapse prevention.4.Paris polyphylla is a hopeful Chinese herb to modulate perfusion and hypoxia. PART TWO Periostin and dentin matrix protein 1(DMP1) are candidate genes for hypoxia and perfusion regulation.Background:Hypoxia is very important for tumor growth and metastasis.Periostin is a new molecule found increased in many cancers.Periostin is a secreted cell adhesion protein of relatively unknown function that has homology with the insect growth cone guidance protein fasciclin-â… .Periostin is thought to function as a homophilic adhesion molecule during bone formation and can support osteoblastic cell line attachment and spreading.Purified recombinant periostin has been shown to be a ligand forα_vβ₃ andα_vβ₅ integrins and proved to promoteα_vβ₃ andα_vβintegrin-dependent cell adhesion and enhance cell motility.Multiple reports have also demonstrated elevated periostin levels in neuroblastoma,epithelial ovarian cancer and in non-small cell lung carcinoma that had undergone epithelial-mesenchymal transformation(EMT) and metastasized.Moreover,it has been shown that periostin potently promotes metastatic growth of colon cancer by augmenting cell survival via the Akt/PKB pathway.Yet, periosin's roles in hypoxia are largely unclear.Dentin matrix protein 1(DMP1) is an acidic phosphoglycoprotein and member of the integrin-binding SIBLING protein family.All of the SIBLINGs have been shown over the years to be up-regulated in many different primary tumors.DMP1, historically thought to be expressed in only bones and teeth,has been shown to be expressed in some normal ductal epithelial tissues recently.Several studies have shown that expression of DMP1 increased in a number of cancerous tissues,including lung,breast,uterus et al.It was reported that there is a coordinate increase in MMP-9 and DMP1 expression in lung and kidney cancer.There is one study shows that DMP1 may accelerate cancer metastasis by bridging MMP-9.Real time RT-PCR super-array was carried out on Lewis cell cultured under hypoxic condition shows that both periostin and DMP1 expression level increased significantly.We hypothesized periostin and DMP1 play an important role in the process of tumor adaptation to hypoxia and low perfusion. PARTâ…¡A If periostin and DMP1 is involved in hypoxic effect on tumor growth?Objectives:1.To address hypoxia's effects on the expression of periostin and DMP1 in rabbit VX2 lung cancer model.2.To identify periosin's effects on tumor growth.Methods:1.The VX2 carcinoma was maintained through serial transplantation into the hind limb muscle of the New Zealand white rabbit.VX2 lung cancer model was established in rabbits by tissue clot suspension injection under CT guidance.2.CT/MRI perfusion scans and blood oxygenation level-dependent(BOLD) MRI were used to assay the oxygen level in tumor.CT guided biopsy were used to get specimen in both hypoxic and non hypoxic area according to the BOLD/perfusion image.3.Total RNA was extracted using Trizle.Real-time RT-PCR was performed for periostin,DMP1,HIF-1αand VEGF.4.HIF-1 expression vectors were transfected to Lewis cells cultured in non-hypoxic conditions.Periostin expression level was assessed by real time RT PCR and ELISA.5.Lewis cells were cultured in hypoxic(1%O₂,5%CO₂) and(21%O₂,5%CO₂) conditions.Periostin expression vector were transected to both group.Methylthiazolete-trazolium(MTT) assay were carried out.Results:1.High BOLD signal,which means low oxygen level,mainly distribute in central area in vivo.mRNA levels of periostin DMP1 in hypoxia area were 4.5 and 3.9 fold in comparison to non hypoxia area.HIF-1 VEGF mRNA levels were also increased in hypoxia area.2.When the VX2 cell from hypoxic area of rabbit tumor cultured in vitro,periostin mRNA level was 6 times higher than that from normoxic area,the periostin protein level higher too.In hypoxic compared to normal condition.DMP1 level was higher than cells from normoxic area.3.Overexpression of HIF-1 in non-hypoxic condition can up-regulate periostin level by about 6 folds compared to pcDNA3 control.DMP1 mRNA level was increased by about 3 folds.Overexpression of HIF-1 increased the relative growth rate of Lewis lung cancer cells,this effect can be blocked partially by periostin neutralize antibody(Figure 4 B,P＜0.05).4.Overexpression of periostin inceased cell relative growth rate in both hypoxic and normoxic condition in Levis cells,but the effect was more significant under hypoxic condition.Conclusion:1.Periostin is responsive to hypoxia and low perfusion.2.Periostin is likely a potent positive regulator of tumor growth in response to hypoxia and possibly a downstream factor of HIF-1.3.DMP1 is another possible molecular in hypoxia regulation process.PARTâ…¡B The possible molecular mechanism in periostin's effect on tumor.Objectives:1.To address how periostin be regulated under hypoxic condition.2.To explore the signal pathway through which periostin exert its effects on tumor growth.Methods:1.Construction of 6.3kb periostin promoter for Dual-Luciferase(?) reporter assay.A.Producing PCR Product from periostin Bac clone using AccuPrime^TM Taq DNA Polymerase.B.Cloning this 6.3kb promoter into pCR(?)2.1 using TA Cloning(?) Kit.Then the plasmid were amplified and collected for digestion. C.Cloning the 6.3kb amplified from pCR(?)2.1into the promoterless pGL3-Basic vector using TA Cloning(?) Kit.pGL3-periostin vector was amplified and purified for later use.2.To observe the effect of TGF-β1on periostin by Dual-Luciferase(?) reporter assay kit.A.pGL3-periostin and pRL-TK plasmid were co-transfect in Levis and VX₂ cells,pGL3-basic and pRL-TK plasmid were co-transfect as negative control.B.TGF-β1protein in serial concentration(2.5,5,10,20ng/ml) was added to the medium 12h after the transfection.C.Promoter activity was assayed by luminometry followed the instruction.3.To observe the effect of TGF-β1 on periostin,recombinant TGF-β1 was added to the culture medium of Lewis cells and real time RT-PCR was carried out.4.To observe the recombinant periostin protein's effect on AKT/PKB signal pathway.A.Preparation of periostin protein.Baculovirus Expression System was used to produce recombinant periostin protein.SF9 insect cell was used in this system.B.The periostin protein in conditioned medium was purified by Heparin Sepharose CL-6B column.The diluted conditioned medium was applied to a Heparin Sepharose column equilibrated with 10 mM phosphate buffer containing 0.15 M NaCl.After the column was washed with the same buffer,proteins were eluted with a step-wise gradient from 0-1500 mM NaCl in 6M urea/Tris-hcl.Western Blot was carried out to confirm the product.C.Levis cell was cultured in 21%O₂ condition with 100 ng/ml of periostin or BSA added.Cell lysates were analyzed by Western blot with the anti-Akt1 /PKB and anti-pS473-Akt1/PKB.D.Periostin expression vector were transected to Lewis cells.Cell lysates were analyzed by Western blot as step C.Results: 1.6.3kb promoter was amplified successfully by PCR.We got 3 clones of bacteria containing 6.3kb periostin-pCR2.1 vector one of them was selected for next step and made into stock.6.3kb periostin-pGL3 vector was built up and confirmed by two different group of restriction endonuclease.2.Periostin promoter relative activity was increased in all the 4 concentrations. There is a dose-effect relation.The periostin mRNA expression levels were increased by 3,5,9 and 11 times respectively by 2.5,5.0,10 and 20ng/ml of TGF-β1.3.Periostin protein was purified from conditioned medium successfully.Western blot shows periostin recombinant protein can increase the phosphorylation of Akt1/PKB on Ser473 significantly.Over expression of periostin has similar effect.Conclusion:1.TGF-β1 can up-regulate periostin expression in vitro and is a possible molecular modulating periostin expression under hypoxic condition.It is a bridge molecular between HIF-1 and periostin2.Periostin can activate Akt1/PKB signal pathway to exert its effect on tumor growth.