Rhein And Emodin Decrease Hypoxia-inducible Factor-1α In Human Pancreatic Cancer Cells And Attenuate Cancer Cachexia In Athymic Mice Carrying The Cells

Objective Normal mammalian cells produce energy primarily by oxidative phosphorylation.Unlike normal mammalian cells,cancer cells produce energy by glycolysis even under the condition of sufficient oxygen.This characteristic of cancer cells is called the Warburg effect.Because glycolysis is of low efficiency in energy production,to produce enough energy,cancer cells over-express glucose transporters and glycolytic enzymes to raise their glycolysis to high levels.It has being known that hypoxia-inducible factor-1alpha(HIF-1α)plays an important role in this process.HIF-1α is an oxygen-sensitive regulative subunit of the transcription factor hypoxia-inducible factor-1(HIF-1).After being synthesized in mammalian cells,HIF-1α is hydroxylated by prolyl hydroxylase proteins-1,-2,and-3(PHDs-1,-2,and-3)and then is degraded in proteasomes.Many tumors,including pancreatic cancer frequently express HIF-1α,and then express HIF-1.This is because the tumors are in hypoxic environment,which can stimulate the production of HIF-1α through oncogene expression and also can inhibit the degradation of HIF-1α.When HIF-1α is expressed,it up-regulates the target genes which will increase the survival ability of cancer cells from the following several aspects: 1.the cancer cells express high levels of glycolytic enzyme and glucose transporter,which will maintain the glycolysis(the Warburg effect)in a high level;2.the proliferation and antiapoptotic ability of cancer cells is enhanced;3.the invasion of cancer cells is enhanced.4.the angiogenesis ability of cancer cells is also enhanced.In addition,the Warburg effect in cancer cells consumes a great deal of blood glucose.In the meantime,lactate is released from cancer cells to the blood as the waste of glycolysis.Then,lactate is converted to glucose in the liver by hepatic gluconeogenesis.After the glucose is released from the liver,it is subjected to tumour glycolysis again.The futile shuttle of glucose and lactate between tumor and liver is called the Cori cycle that greatly increases energy expenditure and hepatic gluconeogenesis and thus increases energy expenditure in cancer patients.As a result,fat and skeletal muscle undergo catabolic metabolisms to mobilize glucose precursors for the liver to maintain its high gluconeogenesis.When such conditions persist,negative nitrogen balance appears and body weight decreases.Then,cancer cachexia occurs.In this sight,HIF-1α promotes the Warburg effect in cancer cells,and in turn the Warburg effect is a triggering event of cancer cachexia.Should HIF-1α expression be inhibited in cancer cells,the Warburg effect would be decreased and then the systemic metabolic disorder induced by Warburg effect may be reversed at least to some extent.We undertook the present study to investigate whether emodin and rhein from Rheum palmatum could inhibit HIF-1α expression in pancreatic cancer cells and also study the relevant molecular mechanism.We also investigated whether the HIF-1α-inhibiting effect of emodin and rhein,if any,attenuate cancer cachexia in the athymic mice carrying human pancreatic cancer Mia Pa Ca2 cells.Method 1.Emodin and rhein were used to incubate five kinds of human pancreatic cancer cells,namely Mia Pa Ca-2,As Pc-1,Bx Pc-3,HPAF-Ⅱand PANC-1.The incubation was made for six hours in a hypoxia environment which contained 1%O2,5%CO2,and 94%N2.HIF-1α expression was determined by Western blotting.We also examined HIF-1α expression when Mia Pa Ca2 cells were exposed to PX-478,noscopine and phenethyl isothiocyanate as they were known to inhibit HIF-1α expression in different cancer cells.Thus,we can observe whether the inhibition of HIF-1α by emodin and rhein is greater than that by the three compounds.2.Mia Pa Ca2 cells were incubated with emodin and rhein in hypoxia condition for six hours.The expression of proteins encoded by HIF-1α target genes were examined by Western blotting including the glycolytic key enzyme,glucose transporter 1,vascular endothelial growth factor and caveolin-1.3.Emodin and rhein were used to incubate Mia Pa Ca2 cells for sixteen hours in normoxia condition(which contain 5%C02 and 95%air)and hypoxia condition.Then,cells migration was examined in Transwell.In the meantime,HIF-1α was knocked out in Mia Pa Ca2 cells by the specific si RNA of HIF-1α in hypoxia condition,the cells migration was also examined. 4.We further explored the molecular mechanisms of HIF-1α expression induced by emodin and rhein in the following four experiments: a.Mia Pa Ca2 cells were incubated by emodin and rhein in hypoxia condition for six hours.Signaling pathway proteins that can stimulate HIF-1α expression were determined by Western blotting.b.Mia Pa Ca2 cells were incubated with emodin and rhein in normoxia for six hours.Culture media were also supplemented with MG132(20μM)to save hydroxylated HIF-1α from degradation.Then,hydroxylated HIF-1α P564 expression was examined by Western blotting.c.Mia Pa Ca2 cells were incubated with emodin and rhein in hypoxia for six hours.HIF-1α m RNA was determined by real-time PCR,and PHD-2 and its m RNA were determined by Western blotting and real-time PCR,respectively.d.Mia Pa Ca2 cells were incubated with normal culture media or media contained emodin or rhein for six hours in hypxia condition.After that,CHX was added immediately to stop protein biosynthesis.Cells were further incubated for 0,10,30,and 60 minutes in hypoxia condition,and HIF-1α expression was determined by Western blotting.HIF-1α degradation rate can be observed.5.Mice were divided into four groups,a group of normal control mice did not inject Mia Pa Ca2 cells,the other groups were carrying Mia Pa Ca2 cells as subcutaneous tumors for eight weeks.In the last four weeks,mice in the three groups were treated with emodin,rhein,or vehicle,respectively,five times a week.Mice body weight,food intake and tumor volume were recorded.When mice were sacrificed,plasma triglycerides were determined.Each tumor was cut in its center along the long axis.Tumor’s maximum section was obtained and stained using a kit of transferase-mediated deoxyuridine triphosphate-biotin nick end labeling.HIF-1α expression signaling pathway proteins that can stimulate HIF-1α expression,and HIF-1 target proteins(including glycolytic enzymes and VEGF)were determined by Western blotting in the tumors.The metabolic changes of liver,fat,and skeletal muscle were determined.Result 1.Emodin and rhein inhibited HIF-1α expression in five kinds of human pancreatic cancer cells.HIF-1α expression was significantly decreased only when PX-478 and noscapine were used in 200μmol/L in Mia Pa Ca2 cells.However,PEITC did not affect HIF-1α expression in the tested concentrations.Thus emodin and rhein had better effect than the three reference HIF-1α inhibiters.2.HIF-1α target proteins including HK-Ⅱ,PFK-1,GLUT1,VEGF,and caveolin-1 were inhibited by emodin and rhein.3.Emodin and rhein could inhibit migration in normoxia,hypoxia and hypoxia with HIF-1α knockout conditions in Mia Pa Ca2 cells.The numbers of migrated cells in hypoxia with HIF-1α knockout condition decreased compared with the wild-type cells in the same hypoxia condition,which means that HIF-1α plays a role in cell migration.4.Emodin and rhein decreased phosphorylated Akt and ERK expression dose-dependently but did not affect total Akt and ERK expression in Mia Pa Ca2 cells.Emodin and rhein decreased hydroxylated HIF-1α expression in normoxia Mia Pa Ca2 cells,thus,emodin and rhein decreased HIF-1α biosynthesis.HIF-1α protein was decreased by emodin and rhein,however,its m RNA was increased by emodin and rhein.The increased m RNA may be secondary to the decrease in HIF-1α protein.Emodin and rhein decreased PHD-2 expression.This result may be also secondary to the decreased HIF-1α protein.When PHD-2 m RNA was determined,it was significantly decreased by emodin but not rhein.Neither emodin nor rhein changed HIF-1α degradation rate.5.Nude mice experimental results showed that: a.emodin could decrease body weight loss in the athymic mice carrying Mia Pa Ca2 cells.b.emodin and rhein inhibited cancer cell’s growth,decreased tumor’s volume and weight,inhibited the expression of HIF-1α and its target proteins,and also decreased two signaling pathways.c.emodin and rein attenuated cancer cachexia in athymic mice based on the liver,muscle and fat metabolism analysis.Conclusion The experimental results showed that emodin and rhein inhibited HIF-1α expression in human pancreatic cancer,and the inhibition of HIF-1α expression reduced cancer cell’s Warburg effect.Emodin and rhein also inhibited cancer cell’s migration.The mechanism of emodin and rhein on HIF-1α was inhibiting its biosynthesis through inhibiting Akt and ERK signaling pathways but not affecting the degradation of HIF-1α.Emodin and rhein also inhibited HIF-1α expression and attenuated cancer cachexia in athymic mice carrying Mia Pa Ca2 cells.