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When treating colon cancer, one of the most common obstacles to the successful treatment is multidrug resistance. For that reason, investigations have been performed to reduce multidrug resistance and help cells escape from cell apoptosis. Still the underlying mechanism is still unclear. In this paper, I will be discussing different recent studies that show advances that could overcome the multidrug resistance in colorectal cancer. According to a recent study the radio sensitizer DCQ (2-benzoyl-3-phenyl 6, 7-dichloroquinoxaline 1, 4-dioxide) enhances sensitivity of HCT116 human colon cancer cells to hypoxia. Though, it is not known whether the p53 or p21 genes influence cellular response to DCQ. We still need to understand the role of these genes in DCQ toxicity and others causes of multidrug resistance.
Keywords: HCT116, multidrug resistance, p21, p53, genes, environmental factors, colon cancer cell lines.
Cancers arise in an area of cells that is predisposed to the development of cancer and it is characterized by a high mutation rate and genomic instability. DNA damages and deficiencies in DNA repair would tend to escape repair and give rise to carcinogenic mutations. Colon cancer is third most common cancer in the United States since it takes 60000 lives per year. Unlike others cancers it is preventable if right screening test are taken on time because it is this disease its clear recognize in the premalignant stage. (Colon Cancer: Current and Emerging Trends in Detection and Treatment). It is the uncontrolled growth of abnormal cells in the body. Cancerous cells are also called malignant. (PubMed Health). This illness arises from mucosal colonic polyps and the two most common histologic types are hyperplastic and adenomatus. The development is a step-wise process that is cause by the accumulation of somatic mutation in the DNA. These mutations are classified as: gain of function events or activation of genes, loss of function events or inactivation of tumor suppressors and epigenetic alterations. The body has mechanisms to identify these deficiencies and repair them. However, if these mutations are too expensive to repair, apoptosis is initiated (Kim). There are several factors that increase the risk of acquiring this sickness which a family history of cancer, familial adenomatous polyposis (FAP) and hereditary nonpolyposis colorectal cancer (HNPCC), which is also known as Lynch syndrome. It is also associated with high-fat, low-fiber diet and red meat. However several studies have found that this risk does not drop if you change to a high-fiber diet. Consequently, this link of diet is not clear. The most common treatments are surgery, chemotherapy and radiation therapy (PubMed Health). Colon cancer patients are at about 9% to 55% risk for development of a second colon cancer in the next 5 years after a first cancer is found, while members of the general population have less than a 1% risk of developing a colonic adenocarcinoma in this period. The human crypt consist of about 10-20 cells at the base of a crypt in an area designated a stem cell niche. With natural selection, a mutant or epigenetically altered stem cell may replace the other stem cells in a crypt, in a process called niche succession. Genetic instability or a mutate phenotype, due to loss of DNA repair or loss of apoptosis competence, would accelerate this process. If, among the stem cells in a colonic crypt, a cell acquires an advantage through a mutation, it will tend to expand clonally at the expense of neighboring stem cells. This process may give rise to "crypt conversion," whereby cells with a mutant or epigenetically altered genotype replace the other cells in the stem cell "niche" and generate an altered genotype for the entire cell population of a colonic crypt. Within a patch, a second mutation or epigenetic alteration may occur. Within this new patch, the process may be repeated multiple times until a malignant stem cell arises which clonally expand into a cancer.
Figure 1 represents diagram of colonic mucosa that indicates the progression of a field defect to colon cancer. The gray area within the right-hand set of irregular concentric areas shows a colon cancer.
Instability in cancer
This cancer has between 49 to 111 non-silent mutations, with an average of 15 of those mutations being "drivers" of carcinogenesis, and the remaining ones being "passengers". It is still unclear on how instability originates. However, it is clear that if an early event in the development of a field defects were loss of DNA repair capability, this would give rise to DNA damages to escape repair which will allow an increased mutations and chromosomes aberrations that are similar to cancer.
According to the study Deficient expression of DNA repair enzymes in early progression to sporadic colon cancer, samples from individual who never had colonic neoplasm were taken to see the expression of Pms2, Ercc1 and Xpf in tissue. These samples were evaluated by immunohistochemistry (IHC) in sequential tissue sections from individuals who never had a colonic neoplasm, and thus are at low risk of colon cancer. The cells of this crypt had high expression of Pms2, Ercc1 and Xpf in the nuclei of most of the cells of the crypts. While Pms2 is expressed at high level in nuclei of absorptive cells throughout the major portion of the crypt, at the open top of the crypt, near the colonic lumen, there is reduced nuclear expression of Pms2. This pattern of expression of Pms2 is typical for crypts within the colonic epithelium of patients at low risk for colon cancer.
A similar pattern of nuclear expression is seen for Xpf in the epithelium of patients at low risk for colon cancer, except that Xpf is sometimes expressed at high levels, and sometimes at low levels, in areas of the colonic epithelium between crypts. The pattern for Ercc1 in low risk patients, however, is to have high nuclear expression in all absorptive cells, both within the crypts and along the epithelium of the colonic lumen.
In figure, we can see the expression of Pms2 (A), Ercc1 (B) and Xpf (C). This crypt is from the biopsy of a 58 year old male patient who never had colonic neoplasia, shows high expression (brown) in absorptive cell nuclei throughout most of the crypt for each of the proteins. A frequent finding was that tissue samples defective in one of the proteins Pms2, Ercc1 or Xpf were simultaneously defective in one or both of the other proteins.
In most cases of reduced expression of Pms2 proteins observed were due to somatic mutation of the Pms2 gene. Somatic mutations in DNA repair genes are infrequent in sporadic cancers, and no Pms2 or Xpf mutations were found in Pms2 and Xpf gene sequences of 11 colon cancer. The results indicated that the substantial deficiencies in protein expression of DNA repair proteins Pms2, Ercc1 and Xpf frequently occur in a coordinated manner in extensive regions, involving as many as 1 million crypts, near cancers, and also occur within cancers suggesting that colon cancers tend to arise in field defects that are deficient in DNA repair and that deficiencies in protein expression of Pms2, Ercc1 and Xpf are frequent early, and often coordinated, steps in progression to colon cancer, as well as further progression within cancers. Our results suggest that blocking any one oncogenic pathway in a colon cancer may only slow down further progression, if the cancer was already deficient in DNA repair. Reduced DNA repair capability combined with increased apoptosis resistance in a cancer, and any metastasized cells from the cancer, would tend to increase DNA damages that give rise to further mutations after DNA replication, likely unleashing other pathways of progression. Our findings also suggest that the current therapeutic approach to treating colon cancer, with surgical removal of the cancer and a good part of the surrounding field defect, is fairly effective, since it would remove many of the secondary and tertiary mutations leading to the cancer. However, this could be followed by use of therapeutic DNA damaging agents. Remaining parts of the field defect (and any metastasized cells) would be more deficient in DNA repair than the surrounding normal areas of the colon, outside the field defect. In addition, it was recently found that inhibition of an additional DNA repair enzyme - such as inhibition of the repair enzyme PARP in breast cancer ââ‚¬" facilitates killing of tumor cells. Similar further DNA repair inhibition in the case of colon cancer could also be pursued. This may cause repair deficient cells in a remaining portion of a field defect, or in metastasized cells, to be even more susceptible to the killing effects of therapeutic DNA damaging agents. (Alexander Facista1)
Multidrug resistance (MDR) is one of the main impediments to the successful treatment of colon cancer. Tumors which obtain resistance to one drug are often resistant to several other drugs as well. One reason for MDR relates to P-glycoprotein (Pgp) and other transporters which are expressed in some cancer cells and could strengthen the efflux of diverse chemotherapeutic agents from cells. Elevated levels of these MDR proteins, which belong to the ATP-binding cassette (ABC) transporter family, strengthen cellular efflux and reduce the effectiveness of anticancer drugs. To conquer drug resistance, inhibitors of MDR proteins have been developed, however their nonspecific inhibition has brought side effects. Glucosylceramide (GCS) can reduce the level of ceramide and allows cellular escape from ceramide-induced cell apoptosis, which has been deemed to be related with MDR. It has been demonstrated that the expression of the GCS gene in drug-resistant K562/AO2 human leukemia cells was higher than that in drug-sensitive K562 cells, and the sensitivity of K562/ AO2 cells to adriamycin was enhanced by GCS inhibition. The mechanisms mediating drug resistance include defective apoptotic signaling and overexpression of antiapoptotic proteins, which regulate apoptotic cell death and which also play an important role in determining the sensitivity of tumor cells to chemotherapy. High level expression of Bcl-2 is found in many human hematologic malignancies and solid tumors.The downregulation of Bcl-2 or other anti-apoptotic proteins, such as Bcl-xL, could either induce apoptosis in cancer cells or could sensitize these cells for chemotherapy. In addition, these proteins protect drug-resistant tumor cells from multiple forms of caspase-dependent apoptosis. Moreover, functional P-gp can inhibit the activation of caspase-3 and-8 by some apoptotic stimuli. Based on the above, we speculate that suppression of GCS by the stable transfection of UGCG shRNA Plasmid would restore sensitivity of multidrug resistance colon cancer cells by the stable transfection of UGCG shRNA Plasmid.
Our results indicated that the mRNA level of GCS in HCT-8/VCR was higher than that in HCT-8, and its level decreased when the HCT-8/VCR were transfected with UGCG shRNA Plasmid. The expression level of MDR1 mRNA was also in the same pattern. This result indicated these two proteins have some relations. We also found that the protein level of caspase-3 was higher in insensitive cells than in sensitive cells. Our research also found that the expression of GCS protein was much higher in HCT-8/VCR than that in HCT-8. And so was the protein level of P-gp. When the HCT-8/VCR was transfected with UGCG shRNA Plasmid, the protein levels of GCS and P-gp were decreased. The results indicated that there may be a relation between GCS and P-gp proteins. Cytotoxity results demonstrated that HCT-8/VCR needs a much higher drug concentration to get 50% inhibition of cell growth. The needed drug concentration decreased when HCT-8/VCR was transfected with UGCG shRNA Plasmid. This result indicated that drug resistance in HCT-8/VCR was reversed. The higher level of the apoptotic gene in the insensitive cells may contribute to the result. Although the drugs can induce apoptosis, the cells with high level GCS may be better able to adapt to the new circumstances, while the sensitive cells may not. The apoptosis rate was higher in insensitive cells than sensitive cells. The result is different with the other researchers. The reason may be the coactions of many apoptotic and anti-apoptotic proteins. In conclusion, our research demonstrated that GCS play an important role in multidrug resistance mechanisms of colon cancer cells with high expression of GCS gene. The up-regulation of GCS could affect the expression of MDR1 in colon cancer cells. They may cooperate with each other in the formation of multidrug resistance (Min Song1)
It is believed that the major causing tumor resistance to radiotherapy and chemotherapy is hypoxia because cause inadequate vascularization in the early tumor development. In the same way, there are several genes that are involved in tumor metastasis and neonangiogenesis that under hypoxia conditions are activated. These hypoxic cells contain high levels of bioreductive enzyme and for that reason represent a therapeutic target to hypoxia-activated drugs. In a recent study conducted by the BioMed Central aimed to investigated the cytotoxicity of DCQ in HCT116 human colorectal cancer cell lines that are either wildtype for p53 and p21 to determine the role of these genes in cellular response to DCQ. It has been previously show that enhanced toxicity in hypoxic tumor cells and as a result they investigate if DCQ causes apoptosis, induce SSB (single strand breaks) and actives the ATM repair pathways in human colon cancer cells. The results of this study indicate that DCQ decreases colon cancer cell grown to great amount under hypoxia. Further studies confirm that higher drug activity under the reducing conditions of a hypoxic environment involved carrying out clonogenic survival assays. Also, DCQ decreased the colony forming ability in a dose-dependent fashion for all three cell lines under both normoxic and hypoxic conditions. They conclude that DCQ is a DNA damaging and apoptotic agent that reduces the viability and colony forming ability of colon cancer and is nontoxic normal intestinal cells. DCQ is a selective cytotoxin in HCT116 human colon cancer cells and its toxicity is independent of p53 and p21. (Mona El-Khatib1)