| Although researchers are keeping to design new experiment model, and try to understand theetiology of cancer, indicate to develop a totally new diagnostic and therapeutic methods, however,cancer incidence and mortality has still not been effectively controlled. The major reason for thissituation is that cancer recurrence still can not be well controlled after chemotherapy, to date,researchers don’t find an effective way to suppress human special drug-resistant tumor recurrence.Chemoresistance might be congenital, which means that part of patients suffer from drug-resistanttumor recurrence even before the start of clinical treatment, in another way, it could also be foundduring the gradual process, and this is a body reaction fighting against the chemotherapy. For example,almost of the non-small cell lung cancer patients who are treated with chemotherapy, they willeventually develop resistance to the use of anticancer drugs. Biological background of chemotherapy isvery complex, in most cases, one or more mechanisms are found during the past researches: inhibitionof apoptosis, induction of human body DNA damage’s repair mechanisms, changing the structures ofrelated clinical drug target, changes in cell membranes (and thereby reduce the absorption of the drug),impactinghe drug efflux pump function, elevating expression levels. Regarding to the matter, a majorreason is the cross-resistance, which is a broad spectrum of cancer cell membrane drug transportersinvolved in, not only for a single active drug but also for multi-drugs Chemical function, as well aschemotherapy agent could be used for curing systemic cancer, even to the administration of the agent tothe patient yet. This phenomenon is known as protein multidrug resistance (MDR), and the proteinsparticipated in this processare called MDR proteins. Multidrug resistance protein study were began in1974, when Victor Ling and Larry Thompson describes a colchicine-resistant cell clones, it was asingle-step selection from stable CHO cell lines, and researchers found that resistant cells did not letcolchicine get into the cytoplasm. The researchers also found that cells resistant to colcine, actinomycinD and vinblastine. It was further discuessed that colchicine sensitive cell uptake function is a passivepathway, resistant cells is followed into an active biological pathway, because drug-resistant cells maybe inhibited by cyanide, inhibited by azide and dinitrophenol. It was further demonstrated that the maindifference between the resistant cells and immature cells is a membrane glycoprotein of170kDa, calledP-glycoprotein expression. P-glycoprotein, It became increasing apparently in a very short timeresponsed way, and it is cery quickly different from the other membrane transport proteins,P-glycoprotein are also involved in several different multidrug resistance mechanisms. For example, itwas described MCF-7/AdrVp (100) resistant cell lines containing a anthracycline95kDa membraneproteinstructure in it in year1990, this protein was names BCRP later(whick means breast cancer resistance protein, also be well known as ABCG2). In1992, Cole, and co-researchers found and clonedcollaboration another phosphoglycoprotein, the enzyme is overexpressed in doxorubicin resistant cellline H69AR, people named this protein as the MRP (Multidrug Resistance-associated Protein). It wassoon the researchers got a clear idea about that all these proteins contain some of the same genesequences and the proteins are involved in functional homology, and are defined as the superfamily ofATP-binding proteins (ATP-binding cassette superfamily, ABCs) members. ATP-binding proteinsuperfamily is the largest family of transmembrane proteins, the whole family contain sevensubfamiliey including A-G, these proteins are based on the similar sequence homologyABCC family members are mainly composed of about200amino acids, this unique amino acidtransmembrane domain of the ABCC family members helps to distinguish ABCC members to othermembers of the ABC. ABCC4is a member of ABCC subfamily (also be well known as the ABCtransporter subfamily C). ABC transporters major transmembrane proteins can mediate theATP-dependent cellular pathways, then t ransport other molecules across the membrane. ABCC4gene islocated on human chromosome13q32.1position, human ABCC gene encoding the expression of theABCC4/MRP, it is the shortest family member, ABCC4protein could mediate endogenous andexogenous molecules’ outflow.In an experiment indicate, ABCC4(also be named as MRP4) could play an importantphysiological role in many biological funcion process, an ABCC4knockout mouse studiesshow, ABCC4could affect liver, brain, and kidney’s function. In cases of cholestatic injuryin the liver, when bile pass through liver, the way is blocked. After bile duct ligation in ratsand mice, ABCC4level increased was observed, and also verify the presence in a patientof familial perihepatic cholestasis. ABCC4plays a role as mediating GSH-dependent bileacid efflux, which is a protective response under certain conditions, the function is toprevent liver from necrosis. In addition to the function of removing metabolites, ABCC4isalso able to resist active drug’s therapeutic effect, protect body from potential damage tovarious other substances may injure significant human tissue. ABCC4deficient mice werefound renal excretory function exhibited when treated by a variety of drugs (anti-cancerdrug topotecan, antiviral drugs, such as be widely used drug, adefovir and tenofovir).Consistent to previous study, Etoposide’s level is elevated in certain tissues and plasma. Lack ofABCC4also mediate topotecan pass through the blood-brain barrier, and then penetrate into the centralnervous system and brain. Furthermore, compared to wild type littermates, ABCC4gene knockout miceare observed a highly drug absorption situation, it shows in bone marrow, thymus, spleen and intestinesfrom ABCC4gene knockout mice. Besides these results, other previous experimental researches have showeded that, ABCC4highly expressed in myeloid progenitor cells, it is transported by active effluxmechanisms, aid to protect these cells from thiopurine induced hematopoietic toxicity. Together, thesestudies highlight the ABCC4play a role as a transporter, it can conduct more than one function, toprotect vital organs and prevent the accumulation of biological metabolites, prevent damage from thechemotherapy drugs.In this study, variety of experimental methodswere used, summarized as follows:93patientsunderwent abdominoperineal resection,28patients got low anterior resection treatment. According topathology reports, it is confirmed circumferential resection margins were negative. After surgery, themedian follow-up time is34.5months(SD=8.6). Then, clinicopathological prognostic factors werestudies. Cox multivariate analysis result showed that the risk of poor prognosis and low3-year overallsurvival rate is related to three types of patients, which are ABCC4expression(P=.036), mutant p53patients (P=.047), pathologic response difference(P=.044); Lentiviral transfected cell lines could beobserved ABCC4protein was decreased. Real-time PCR and Western Blot results were confirmed abcc4gene expression was inhibited in RKO-KD, HT29-KD cells, this model provide a theoretical basis forthe subsequent cell function test, it helps continue to explore the molecular mechanisms study research.Real-time PCR showed abcc4mRNA expression levels in HT29-KD group (0.20±0.02) wassignificantly less than HT29-NC group (0.99±0.02) and HT29-CON group (0.98±0.03)(P <0.05);Western Blot experiments suggest ABCC4protein expression in HT29-KD group (0.25±0.02) wassignificantly lower than HT29-NC group (0.98±0.02) and HT29-CON group (0.98±0.03)(P <0.05).Between the negative control group HT29-NC and blank control group HT29-CON reseach, there is nosignificant difference between negative control group HT29-NC and blank control group HT29-CON(P>0.05). Calculated by measuring the size of tumor tissue, we found that compared with the controlgroup, RKO-KD xenograft model of tumor growth in a time-dependent state after radiotherapy, thetumor growth was significantly inhibited (P <.05), measurement of the average tumor weight, RKO-KDtumor weight2.01g (±0.18) was significantly lighter than RKO-NC group3.97g (±0.21) andRKO-CON group4.01g (±0.19)(P <.05). Among these three groups, the control group’s tumor size innude mice was not statistically significantly different, the weight was not statistically significantlydifferent either(P>.05). After4Gy dose irradiation treatment, the rapid rise in intracellular cAMP levelswas observed, showing this effect is folloew by time-dependent change. The average concentration ofcAMP in RKO-KD Group is0.66(±0.25)%compared RKO-NC group0.45(±0.15)%and RKO-CONgroup0.46(±0.16)%, the average concentration of cAMP in RKO-KD Group was significantly higher(P <.05). After same treatment, cell cycle distribution was detected,43.54(±3.07)%proportion in theG0/G1phase of the RKO-KD cells was significantly less than the RKO-NC group’ value65.73(± 3.78)%and RKO-CON group66.52(±3.64)%(P <.05), indicating that RKO-KD cells lose G1-S DNArepair capacity, monitoring points’ function is effected, when cancer cells were proceeded into arelatively radiation-sensitive G2phase, the cancer cells were more vulnerable to radiation damage. Inthree blank groups with non-irradiated treatment, the cell cycle distribution was not found significantstatistically difference (P>.05). In summary, the results may suggest a hypothesis, MPR4overexpression may cause radiation resistance, after reducing the expression of MRP4, then radiotherapymay increase the apoptosis rate of tumor cells. |
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