| β-elemene, the active antitumor component of elemene, has recently been demon strated to enhance the radiosensitivity of human cancer cell lines in vitro and of one animal tumor in vivo. However, the underlying mechanism is still unclear, and there are many unkown areas merit further investigation.Hypoxia is a characteristic feature of tumor microenvironment and solid tumors are dependent on the transcription and translation of many hypoxia-ralated genes to adapt to the hypoxic environment.The best explored transcription factor induced in hypoxic cells is HIF-1,which is composed of anαsubunit and aβsubunit. The expression of HIF-1αis regulated by oxygen and HIF-1βis constitutively expressed. Under hypoxic conditions, HIF-1αhas been found to directly bind to the survivin promoter and to positively regulate the transcriptional activity of survivin. Human cancers with high expression levels of HIF-1αor survivin exhibit increased resistance to radiotherapy and are associated with higher risk of local recurrence and poor overall survival. Therefore, inhibition of survivin or HIF-1αmay be a promising way to enhance tumor radiosensitivity. One of the main regulation mechanisms of HIF-1αis by PI3K/AKT/mTOR pathway, which has close relationship with resistance to radiotherapy. Moreover, the expression of survivin is recently found to be regulated by mTOR pathway.Previous studies have attributed the enhanced radiation response to increased apoptosis and cell G2/M arrest,and survivin is just regarded as a key regulator of cell division and inhibition of apoptosis.HIF-1α,which has close relationship with hypoxic condition of tumor,is the key transcriptional factor of survivin. Whetherβ-elemene plays its radiaosensitizing role by inhibiting expression of HIF-1α/survivin or PI3K pathway to regulate the expression of HIF-1αto amliorate hypoxic condition is not been published so far.β-ele mene is a novel drug that acts on mult-targets, so it is a feasible method to find new target by high throughput proteomics technology.Objective:1.The nude mice model transplanted lung adenocarcinoma A549 cell line was successfully established, the growth inhibition of tumors by different dose ofβ-elemene was observed,and optimal radiosensitive dose was obtained. Further, the effect ofβ-elemene combined with radiation on endogenous and exogenous hypoxic marker-CAⅨand pimonidazole was studied.2.The correlation of the radiosensitizing effect ofβ-elemene with HIF- 1α,survivin or apoptosis was studied. Further,the association betweenβ- elemene and PI3K/mTOR pathway was also been investigated. 3.For screening valuable novel targets ofβ-elemene, the two-dimensional differential in-gel electrophoresis technology was used to find differential proteins between radiation group andβ-elemene+radiation group .And then the novel target was verified by using RT-PCR and Western blot.Methods:Part one1.The transplanted mice model was established through the cell suspension inoculation. The transplanted mice with 0.8~1.0cm~3 tumor were randomized(5 per group) and treated with Nacl,β-elemene at 25,45 or 100mg/kg dose or local tumor radiation at a single dose of 5Gy or com bination therapy ofβ-elemene and radiation. Radiation treatments were performed 1 hour afterβ-elemene was injected intraperitoneally.Radiation was delivered by 6 MeV electron beams from a linear accelerator (Varian) at a single dose of 5 Gy.2.The tumor sizes were measured every 2 days using Vernier calipers. At 4-5 weeks, tumor volumes reached the required size (0.8~1.0 cm~3). The average tumor inhibition rate (ATIR) was calculated every day until tumors in the control group doubled in size. The activity ofβ-elemene to enhance tumor radioresponse was evaluated by EF, the Enhancement Factor. The optimal radiosensitizing dose was selected for later experiments based on EF calculations.3.The new transplanted mice with 0.8~1.0cm~3 tumor were rando mized(5 per group) and treated with Nacl,β-elemene at optimal radio sensitizing dose, local tumor radiation at a single dose of 5Gy or com bination therapy ofβ-elemene and radiation. Mice were sacrificed and the tumors were excised 24 hour after treatment. 4.The expression of CAⅨand pimonidazole hydrochloride was investigated by immunohistochemistry.Part two1.The mRNA expression of HIF-1α,survivin,PI3K and mTOR in every group was investigated by using RT-PCR.2.The protein expression of HIF-1α,survivin in every group was investigated by using Western blot.3.Immunohistochemical staining of HIF-1α,survivin in every group was investigated.4.The apoptosis in every group was tested by TUNEL staining.Part three1.The differential proteins between radiation group andβ-elemene+radiation group were screened by the two-dimensional differential in-gel electrophoresis technology.2.The expression of peroxiredoxin-1(Prx-1) was tested in every group by RT-PCR and Western blot.Results: Part one1. The nude mice model was successfully established and the growth curves was drawed.The growth curve of each experiment group was obtained. Within the time range of 2~8 days, the variation tendency of the average tumor inhibition rate was consistent with the volume change inβ-elemene groups at 25,45 or 100mg/kg dose. The antitumor activity ofβ-elemene was in a dose-dependent manner.2. The EFs of 25 mg/kg, 45 mg/kg and 100 mg/kg dose were 0.84, 1.24, and 2.04 respectively.Since the drug is considered to exhibit synergistic effect with radiation only at a dose level where EF is greater than 1, this suggested that there was a synergistic effect when at least 45 mg/kgβ-elemene was used with radiation therapy.This dose level was subsequently selected for later experiments. The 100 mg/kg dose level was not chosen, in order to minimize the direct cytotoxic effect ofβ-elemene. 2.Expression of CAⅨ: Compared with control group, the expression inβ-elemene group was increased(p<0.05),but the expression in radiation group was slightly increased(p>0.05),.However,the expression in combined group was significantly decreased compared with the other group (p<0.05).3.Expression of pimonidazole hydrochloride:Compared with control group, the expression inβ-elemene group had no obvious difference(p>0.05),but the expression in radiation group was significantly increased(p<0.05). However,the expression in combined group was significantly decreased compared with the other groups (p<0.01).Part two1.Expression of HIF-1αand survivin: HIF-1αand survivin mRNA were increased in theβ-elemene group as compared with the control group (HIF-1α,p<0.01;survivin,p<0.05); HIF-1αand survivin proteins were decreased in theβ-elemene group as compared with the control group (p<0.05).The mRNA and protein levels were both higher in the radiation group when compared to the control group(p<0.01).The mRNA and protein levels were both significantly decreased in theβ-elemene/radiation cotreat ment group (p<0.01). The immunohistochemistrial staining results are showed:β-elemene alone had little effect on survivin and HIF-1αexpression compared with the control group(p>0.05). Radiation alone significantly increased both survivin(p<0.01) and HIF-1α(p<0.05) compared with the control group. By contrast, whenβ-elemene was used together with radiation, expression levels of survivin and HIF-1αwere significantly lower than in the control group (p<0.01).2.Results of TUNEL:there were higher levels of apoptosis in the radiation group than in the control group (p<0.05). However, there were still higher levels of apoptosis in tumor samples treated with bothβ-elemene and radiation as compared to either of the monotherapies (p<0.05). More apoptotic cells were also detected inβ-elemene treated tumors than in the control group (p<0.05).3.Expression of PI3K,mTOR mRNA:PI3KmRNA was no obvious changed in theβ-elemene group as compared with the control group (p>0.05). But PI3KmRNA was significantly increased in the radiation group compared with the control group (p<0.01). PI3KmRNA was no obvious changed in the combined group as compared with the radiation group (p>0.05).However, the levels inβ-elemene/radiation group were signifi cantly decreased as compared with the control group(p<0.01).The expres sion of mTOR mRNA was not detected in the combined group.Part three1.The DIGE images were analysed by software and there were 17 protein spots were discoveried as differential proteins. The expression of 8 protein spots were decreased and 9 protein spots were increased in combined group as compared with radiation group.2.The differential proteins were analysed by mass spectrometry and retrieved by databases.A radiation-related protein(NO.780)-peroxiredoxin -1(Prx-1) was found.3.Expression of Prx-1 mRNA:Prx-1mRNA was significantly decreased in theβ-elemene group, increased in the radiation group compared with the control group(p<0.01). Prx-1mRNA was significantly decreased in the combined group compared with the control group(p<0.01) and also significantly lower than theβ-elemene group(p<0.01).4.Expression of Prx-1 protein:Prx-1 protein was no obvious change in theβ-elemene group compared with the control group(p>0.05). Prx-1 protein was significantly increased in the radiation group, decreased in the combined group compared with the control group(p<0.01).Conclusion:Part one1. The nude mice model was successfully established and tumor regres sion and regrowth were monitored. The antitumor activity ofβ-elemene was in a dose-dependent manner.2. The tumor volume doubling times in each group were obtained to calculate the EFs of respective drug doses. 45mg/ kg was selected for later experiments at last.3.β-elemene at radiosensitizing dose improved the expression of CAⅨand had little effect on the expression of pimonidazole hydrochloride.The results suggested thatβ-elemene alone at radiosensitizing dose did not amliorate tumor hypoxic condition.4. Radiation significantly induced both of the two markers that suggested the hypoxic condiction of tumor become serious 24 hours after 5Gy radiation and improved the radioresistant.5.β-elemene combined with radiation significantly inhibited endoge nous and exogenous hypoxic marker-CAⅨand pimonidazole hydrochlo ride.The results suggestedβ-elemene enhanced tumor radioresponse by amliorating tumor hypoxic condition.Part two1.β-elemene at radiosensitizing dose combined with radiation signifi cantly inhibited the mRNA and protein expression of HIF-1αand survivin. The results suggested HIF-1αand survivin were new targets ofβ-elemene.2.β-elemene at radiosensitizing dose combined with radiation signifi cantly induced apoptosis of xenografts.The results suggestedβ-elemene induced tumor apoptosis by inhibiting HIF-1α-survivin pathway. 3.β-elemene at radiosensitizing dose had no effect on the expression of PI3KmRNA,but significantly inhibited the expression of mTOR mRNA.β-elemene combined with radiation significantly inhibited the expression of mTOR mRNA,that suggested mTOR was a potential target ofβ-elemene.4.Radiation induced the mRNA and protein expression of HIF-1αand survivin,but significantly inhibited the expression of mTOR mRNA. The results suggested the main reason that radiation induced the expression of HIF-1αand survivin is related to reactive oxygen generated by radiotherapy that leads to defensive reaction of tumor and not to mTOR pathway.Part three1.There were 17 protein spots were discoveried as differential proteins by the two-dimensional differential in-gel electrophoresis technology. The expression of 8 protein spots were decreased and 9 protein spots were increased in combined group as compared with radiation group. The differential proteins were analysed by mass spectrometry and retrieved by databases.A radiation-related protein(NO. 780)- peroxiredoxin-1(Prx-1) was found.2.Compared with the control group, Prx-1 mRNA was significantly decreased in theβ-elemene group,but Prx-1 protein was no obvious change.The results maybe dues to post-transcription mechanism. Compared with the control group, Prx-1 mRNA and protein were significantly increased,that suggested radiation induced the expression of Prx-1.3. Compared with the control group, Prx-1 mRNA and protein were significantly decreased in combined grou,that suggested that Prx-1 was novel target ofβ-elemene... |
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