Study On The Effect And Mechanism Of Curcumin Increasing Sensitivity Of Chemotherapy In Laryngeal Carcinoma Cell Hep-2

Laryngeal cancer as the second high incidence of respiratory tractcancer which is incidence increased year by year. At present, the maintreatment for laryngeal squamous cell carcinoma can be divided intothree types: surgery, radiotherapy and chemotherapy, chemotherapy aslaryngeal squamous cell carcinoma and the main treatment of other headand neck squamous cell carcinomas. But the carcinoma which isaforementioned often has types of treatment barriers like insensitive tochemotherapy, chemotherapy resistance and easy to relapse afterchemotherapy. Therefore, how to enhance the laryngeal cancerchemotherapy sensitivity, reduce the resistance effect of chemotherapy,became one of the highlights in agro-scientific research which is thebroad scholars fighting for.Scholars found that it is easy to generation multi-resistant laryngealcancer cells during the chemotherapy,which reduce tumor sensitivity tochemotherapy and enhancing the resistance. People also discoveredanother thinking to explain the cancer chemotherapy is not sensitive, "cancer stem cell theory:" The resistance to chemotherapy can beachieved by cancer stem cells, chemotherapy can kill tumor most cellsand has no effect on cancer stem cells, tumor stem cell proliferation, andcan eventually lead to tumor recurrence. As a result, improve thesensitivity of tumor cells to chemotherapy drugs, reversal cancer stemcells to chemotherapy resistance, has become the key to improve theeffect of tumor treatment in current.Curcumin is plant polyphenols which is a kind with yellowcrystalline powder, derived from Curcuma rhizomes. Curcumin canaffect anti-inflammatory, antioxidant, anticoagulation, antitumor,resistance mutation etc, especially in specific inhibition CSC. Previousstudies have confirmed that curcumin have obvious inhibitory effect onprostate cancer cells, human nasopharyngeal carcinoma cells and gastriccancer cells, human colon cancer cells and human liver cancer cells.Butthe effect of curcumin on laryngeal cancer cell are rarely reported, inaddition,the mechanism of such effect is still unclear. Therefore, ourresearch will explore the influence of curcumin on laryngeal cancer cellbiology behavior, explore the curcumin and cisplatin on the anti-tumoreffect of laryngeal cancer cells, enhance the mechanism of the effect thatcurcumin can improve chemotherapy sensitivity laryngeal cancer cells toanalysis of. The application of curcumin in laryngeal cancerchemotherapy provide the theoretical basis, as well as the specific role of curcumin in cancer stem cells to provide theory support, for the finallooking for new way to improve the clinical effect of laryngeal cancerchemotherapyResearch methods:In order to explore the sensitization effect of curcumin for laryngealcancer, our experiment is divided into two parts of the in vitro and invivo, and take the following methods:In vitro experiment:1. Colony forming experiment, MTT experiment-curcumin combinedcisplatin for laryngeal cancer Hep-2the effects of cell proliferation2.Annexin-V/PIDouble standard experiment-curcumin combinedcisplatin for laryngeal cancer Hep2cells apoptosis3. Cell scratch experiment-curcumin combined cisplatin for laryngealcancer Hep2cells in vitro migration4. Flow cytometry instrument test and flow cytometry instrumentCD133+cells separation experiment-curcumin and cisplatin to Hep2cells of CD133+cells5.Western-blotExperiment-turmeric’s influence on the ABCG2expression of CD133+cellsIn vivo experiment：1. Determination of tumor growth2. Cancer terminal volume measurement 3. Tunel method-the role of curcumin and cisplatin on tumor cellapoptosis4. Western blot method, detecting the expression of tumor apoptosisrelated proteinsresult：In vitro experiment：1. Colony forming experiment shows that the combined use of cisplatinand curcumin can reduce Hep2cells proliferation, inhibition colonyformation, untreated group of clonal colony (greater than100cells)for526±35, curcumin and cisplatin group formed colony numberdecreases, the colony count of curcumin in combination withcisplatin group was obviously reduced to150±40, and the size of thecolony also decreased significantly, P <0.05, the difference isstatistically significant;Pure curcumin and curcumin liposomesgroup was no significant difference, which also confirmed thatliposomes can be used as the carrier of curcumin and can not effectthe throat can’t Hep2cells to the biologyobviously.2. It is further confirmed that the combined application of curcuminand cisplatin can significantly inhibit the Hep2cells proliferationwhich determined by MTT experiments.Single application ofcisplatin group acheive to reduce proliferation by around60%, whilein the curcumin combined cisplatin dosage group, do around90%, P <0.05, the difference is statistically significant.Also, the experimentproved that the liposome can be used as a carrier of the security, toHep2cells。3. Annexin V/PE double standard method experiments confirmed thatcurcumin and cisplatin to Hep2cells separately for48hours, canpromote apoptosis of Hep2cells when application of curcumin andcisplatin plus, this to promote the role of apoptosis is moresignificant.Untreated group of Hep2cells apoptosis percentage was2.35±1.6%, the individual treatment of curcumin and cisplatin groupof Hep2cells apoptosis percentage increases to13.4±2.8%and32.1±9.5%, and curcumin+cisplatin plus medication group of Hep2cells apoptosis percentage is increased dramatically,54.4±11.8%.4. Scratch test of curcumin and cisplatin for laryngeal cancer Hep2cells in vitro migration: the curcumin group, cisplatin group andcurcumin+cisplatin group of Hep2cells compared with blankcontrol group migration distance shortened, and the joint drug groupof Hep2cells migration distance shows more apparent, this suggeststhat curcumin combined cisplatin for laryngeal cancer Hep2cells invitro migration ability has obvious inhibitory effect.5. Flow cytometry instrument test and flow cytometry instrumentCD133+cells sorting experimental detection: untreated group ofHep2cells, CD133+cells of the average detection rate was4.5 ±1.2%, cisplatin group of Hep2cells, CD133+cells detection ratewas6.89±1.1%, but the cisplatin plus curcumin combined druggroup of Hep2cells, the CD133+cells detection rate decreasedsignificantly,1.49±1.0%.Cisplatin group compared with cisplatin+curcumin combined dosage group has obvious peak curve shift tothe left, the results show that cisplatin can make effective CD133+concentration, when cisplatin combined application with curcumin,the enrichment is obvious inhibition.6. Using western blot method to detect the ABCG2protein expressionin CD133+cells, CD133+shows high expression of ABCG2inuntreated group, in curcumin+cisplatin group shows lowerexpression;In CD133-cells, ABCG2also show lower expression,ascomparison of beta action.Visible, after packet processing, CD133+and CD133-cells were collected and detection of ABCG2proteinexpression, unprocessed and cisplatin treatment group was nosignificant difference in ABCG2expression of CD133+cells, and incurcumin+cisplatin plus medication group, ABCG2expression ofCD133+cells with untreated group and CD133-cells in theexpression of ABCG2, for lower expression.In vivo experiment：1. Tumor growth curve shows: since6days after delivery, curcumin+cisplatin group tumors had less than the control group (P=0.014), starting from9days after delivery, curcumin+cisplatin groupstarted less size than pure curcumin group of tumor(P=0.026), andstarting from12days after the treatment, tumor volume of curcumin+cisplatin group had less size than cisplatin group (P=0.043), andthe differenceincrease with the drug delivery time extension, thecontrol of tumor volume compared with curcumin group, nosignificant difference.The above results suggest that curcuminsignificantly improved the laryngeal cancer chemotherapysensitivity.2. Terminal of tumor volume measurements: cisplatin+curcumin andcisplatin group of tumor volume compared with blank control group,the curcumin group reduced, cisplatin+curcumin group terminaltumor volume decreased more obvious.3. Tunel staining the experiment to detect the apoptosis of tumor cells,the results showed: in the blank control group and the curcumingroup, the percentage of Tunel positive cells were24.4±6.2%and25.2±8.8%, no significant difference;In cisplatin group, thepercentage of Tunel positive cells was38.6±7.2%, while in curcumin+cisplatin group, the proportion of positive cells was59.6±6.9%,the percentage of Tunel positive cells, the cisplatin group increasedsignificantly, suggesting that curcumin effectively improves theapoptosis occurred after laryngeal cancer chemotherapy.This result is consistent with the experimental results in vitro.4. Western blot experiments to detect apoptosis related proteinsexpression levels of caspase3: in the blank control group and thecurcumin group, caspase-3protein expression level had nosignificant difference;In the chemotherapy group, the expression ofcasepase-3, in the expression of curcumin+cisplatin group weresignificantly higher than the other three groups (P <0.05), consistentwith Tunel staining results, further affirmation of curcumin doesincrease the chemotherapy cancer tissue apoptosis.Conclusion:1. cisplatin inhibits proliferation, migration of larynx Hep-2cells, andpromote the apoptosis of larynx Hep-2, Joint application ofcurcumin can improve the chemotherapy sensitivity of larynxHep-2cells.2. A few of CD133+cells wich exist in Larynx Hep-2cells canresistance chemotherapy, curcumin can inhibition the concentrationof Hep-2cells of CD133+cells in the chemotherapy, reverse thelarynx Hep-2cells of CD133+cells in chemotherapy resistance byreducing the expression of ABCG2protein.