The Anti-cancer Effects And Underlying Mechanisms Of Chemical Modified Tetracyclin3

Background:Cancer has became one of the leading causes of death. Although the combinedstrategies of chemotherapy, surgery and radiotherapy increased the survival rate ofcancer patients, the rapid proliferation and metastasis remained the major causes ofdeath. Thus, the identification of new anti-cancer agents and the strategies based on theeffective combinational approaches are necessarily developed. Statistically, cervicalcancer is currently the second most common malignant disease in women all over theworld, and hepatocellular carcinoma (HCC) is reported to be the fifth most diagnosedcancer worldwide.Chemically modified tetracyclines (CMTs) are the analogs of tetracycline. For theretained character of matrix metalloproteinases (MMPs) inhibitory property, CMTs areinvestigated as the potential anti-cancer drugs. According to pervious studies, CMT-3was recognized to be the most promising anti-tumor compound among CMTs. Theability to inhibit proliferation and invasion of various cancers of CMT-3have beenconvinced by numerous in vivo and in vitro researches. Furthermore, CMT-3wasevaluated in preclinical cancer models and had entered early clinical trials in patientswith malignant diseases. However, the adverse effects of CMT-3(e.g. nausea, vomiting,diarrhea, mucositis, leukopenia, and thrombocytopenia) were observed in clinical trialswhich limited its application. According to previous studies, a large number oftraditional Chinese herbs (e.g. Garlic, mistletoe, curcumin, resveratrol, silibinin,berberine, quercetin, and celastrol) possessed beneficial effects not only on anti-cancerprogression, but also on the promotion of apoptosis and alleviation of toxicities inducedby chemotherapy chemicals. Similarly, Tanshinone IIA sodium sulfonate (TSN-SS), apopular Chinese cardiovascular medicine, has been proved to have antioxidantproperties and cytotoxic activity against multiple human cancer cell lines.However, the underlying mechanism of CMT-3alone and its combination withTSN-SS on growth and metastasis of cancer cells are not studied. Further investigation on these mechanisms will provide sufficient basics for CMT-3serving as a potentialtherapeutic strategy against human cancer, and TSN-SS serving as a novelchemo-therapeutic adjuvant.Objectives:The aim of this project is to investigate the underlying mechanisms (e.g. apoptosis,cell cycle arrest) by which CMT-3exhibits its inhibitory impact on cancer in vitro andvivo. In addition, the novel mechanisms (related to HMGB1release and autophagy)were studied in a combinational effect of CMT-3and TSN-SS.Methods:For in vitro studies, MTT and clonal formation assay were used to test cell viabilityand cell growth; Alteration of cells morphology was observed by the Cell R Live CellStation. HeLa and Siha cells were stained by CellTrackerTM Green CMFDA (5μM) orHoechst33258(5μg/ml) before observation; For testing the autophagosomes, HepG2cells were stably transfected with GFP-LC3and obesrved by fluorescence microscope;Cells migration and invasion were studied by scratch healing and trans-well assays; Theactivity of matrix metalloproteinases (MMPs) was examined by Gelatin Zymographymethod. The ROS content was measured by a ROS detection kit; Apoptosis inductionwere tested using an Annexin V/PI for double staining kit; The cell cycle distributionwas tested on the Flow Cytometry system; The related protein expression weremeasured by Western blot assay; mRNA expression was determined usingsemi-quantitative RT-PCR assays; The location and expression of protein was detectedby immunofluorescence analysis.For in vivo studies, the Babl/c nude mice model with trasplanting human cervicalcancer HeLa cells were established and assigned to following groups randomly. The1stgroup was given saline solution as negative control; The2nd group was given cisplatinas positive control (5mg/kg,1/week, ip); The3rd or4th group was given1%sodiumcarboxymethyl cellulose (CMC,1/4days, ig) or nanostructured lipid carrier (NLC,1/4days, ip) without CMT-3; The5th group was given CMT-3dissolved in1%CMC(20mg/kg,1/4days, ig); The6th group was given CMT-3which loaded NLC (6mg/kg,1/4days, ig). The tumor volume was measured every2days up to total28days, and themice weights were measured at the end of this experiment. The ultrastructure in tumortissues were observed by Technique of Biological Electron Microscope. Results:The data from MTT assay showed that CMT-3inhibited the growth of variouscancer cells in a dose-and time-dependent manner. But there was a cell-type differenceamong these cell lines. For example, compared to breast cancer, liver and lung cancercell lines are seemed to be insensitive to CMT-3. On the other hand, CMT-3sensitivitywas also related to the status of the p53gene. In the cancer cells originated from thesame tissue, p53wild-type cell lines were relatively more sensitive to p53mutant celllines. In contrast to clinical conventional chemo-therapy drugs, the inhibitive ability ofCMT-3was better than cisplatin and pirarubicin hydrochloride in human cervical cancerHeLa cells and human hepatocellular carcinoma HepG2cells, and even better than thatof Taxol in human breast cancer MCF-7cells. Clonal growth assays indicated thatCMT-3significantly inhibited growth of breast cancer MCF-7cells and HepG2livercancer cells, but a reduced effect of CMT-3was observed when it combined with PTF-α(P53inhibitor). In addition, CMT-3significantly inhibited the adhesion, migration, andinvasion in human cervical cancer HeLa cells, and the expression and activity ofMMP-2and MMP-9were decreased by CMT-3.CMT-3induced HeLa cells shrink, turn round, more loosely arranged and adhered,at last the majority of cells were floated in the nutrient medium. But, Siha cells wereless sensitive to CMT-3compared to HeLa cells. CMT-3induced intracellular ROSlevels in a dose-and time-dependent manner in HeLa cells, accompanied by theincreased level of apoptosis. However, the inhibitor of ROS, NAC, significantly reducedthe increased apoptosis induction induced by CMT-3. Furthermore, the expression ofLC3-I and LC3-II was obviously increased in HeLa cells treated with CMT-3. Even incombination with saturated concentration of autophagy inhibitor, BafA1, CMT-3stillstimulated the increased expression of LC3-II. In Siha cells, CMT-3promoted G0/G1phase cell cycle arrest instead of apoptosis. The anti-cancer effects in HeLa cells wasrelated to mitochondria-dependent signaling, increasing apoptosis related proteinsexpression level (cleaved caspase-9, cleaved caspase-3, cleaved PARP) but decreasingthe expression of pro-caspase-9, pro-caspase3, PARP, Bcl-2and NF-κB. The G0/G1phase cell cycle arrest in Siha cells was related to the expression of Cyclin E instead ofits transcription, and a translocation of NF-κB from cytoplasm to the nucleus. Inaddition, MTT and clonal formation experiment suggested that CMT-3had nosignificant effect to HeLa cell survival rate in combination with X-ray, compared to X-ray treated alone.In animal experiment, under the condition of giving20mg/kg CMT-3orally, themice expressed certain side effects, including losing weight and mild anxiety reaction.However, when giving6mg/kg CMT-3through intraperitoneal injection, the mice hadno significant side effects. The transplanted tumors were obviously inhibited by theapplication of CMT-3(orally or intraperitoneal injection), and even better thantraditional chemotherapy drug, cisplatin. Technique of biological electron microscopedemonstrated that apoptosis and autophagy were ongoing after the treatment of CMT-3.In HepG2cells, CMT-3increased autophagosome synthesis, rather than inhibitedLC3-II degradation when co-treatment with bafilomycin A1at the saturatingconcentrations. Moreover, CMT-3stimulated HMGB1release to medium. Theco-treatment of autophagy inhibitors (CQ,3-MA and Baf A1) with CMT-3promoted theaccumulation of cytoplasmic HMGB1; Under the condition of co-treatment with CMT-3and TSN-SS, the growth of HepG2cells was inhibited excessively compared to CMT-3treated alone. Furthermore, in the additional presence of100μM TSN-SS, the intensityof LC3-II was dramatically increased, which suggested that TSN-SS facilitated theautophagy level. In contrast to HMGB1releasing to cytoplasm after CMT-3incubation,additional treatment of TSN-SS inhibited HMGB1release, and it trapped HMGB1locating in nuleus. Similarly, after co-treatment with50,100μM TSN-SS, thecytoplasmic HMGB1level was dramatically decreased compared to5μM CMT-3incubation alone, but the total intracelluar HMGB1was increased in the presence of50,100μM TSN-SS, confirming that TSN-SS could indeed inhibit HMGB1release inCMT-3treated HepG2cells.Conclusions:In conclusion, the presented study indicates that CMT-3inhibits the growth andproliferation of different types of cancer cells in vitro and vivo. Different cancer cellshave varying sensitivities to CMT-3, which possibly related to the status of p53. Theintrinsic mitochondrial damage induced by oxidative stress might play an important rolein CMT-3cytotoxicity. Also, NF-κB may play as a key mediator for the response ofcervical cancer cells to CMT-3. CMT-3had no radio-sensitive effect when co-treatmentwith different dose of X-ray in HeLa cells. CMT-3promotes the level of autophagy andHMGB1release in HepG2cells, and autophagy could be one of the pathways ofHMGB1degradation. In addition, TSN-SS facilitates CMT-3’s cytotoxicity effect in HepG2cells. The underlying mechanisms including the promotion of autophagy butinhibition of HMGB1release are demonstrated. These findings provides the novelmechanism underlying CMT-3modulated cancer cell extermination effect, which maypave the road for developing new candidates for clinical cancer chemotherapy. It alsoprovides sufficient basics for TSN-SS serving as a novel chemo-therapeutic sensitizer.Further study should be conducted on the improvement of cancer cells cytotoxic effectsand reduction of adverse effects to normal tissue, including search the novel carrier ofCMT-3and its sensitizers.