The Expression And Significance Of GRP78 And Calreticulin In Human Osteosarcoma

BackgroundOsteosarcoma is a primary malignant tumor of the skeleton, which frequently occurs in adolescent. Various therapeutic tools failed in radical cure, due to the character of infiltrating growth in Osteosarcoma. Before 1970, all patients with Osteosarcoma were treated by amputation but the 5-year survival rate was under 10% and almost all patients died within a year from diagnosis. Dramatic therapeutic improvement achieved in the last 30 years is a result of development of aggressive and efficient combination chemotherapy regimens. Although neoadjuvant chemotherapy is effective in improving patient survival, the frequent acquisition of drugresistant phenotypes and the occurrence of adverse reaction are often associated with chemotherapy, which remain as serious problems. Therefore, the treatment of osteosarcoma is placed at a new stage and there is a clear need for novel biomarker which could be used to determine the characteristic and prognosis of osteosarcoma, and could be applied as therapeutic targets for gene therapy of osteosarcoma. Glucose-regulated protein 78 (GRP78), also refered to as Bip(immunoglobulin heavy-chain binding protein, is a major endoplasmic reticulum chaperone with Ca²⁺-binding and antiapoptotic properties. In recent years, several studies have shown that GRP78 was overexpressed in multiple tumor types, which was correlated with chemoresistant property and antigen expression of the tumor cells. Furthermore, several studies have indicated that GRP78 can promote tumor cell proliferation, survival, metastasis, and resistance to a wide variaty of therapies. GRP78 could also inhibit apoptosis through mediating signal transduction pathways, such as suppressing the signal cascade of PKC-ε/ERK/AP-1, inhibiting the activation of caspase-7 and Bax. In the micro-environment of solid tumor, the expression of GRP78 could resist the cytotoxicity effect on G1 and S phase of the chemotherapeutics and block the apoptosis effector, which could inhibit apoptosis. Recent studies have confirmed that GRP78 could be applised as a molecular target to promote apoptosis, and to overcome the drug-resistance of cancer cells. Therefore, combination therapy suppressing GRP78 expression may represent a novel approach toward eradication of residual tumors.Calreticulin (CRT) is also an endoplasmic reticulum protein, with some unique features, including regulation of Ca²⁺ homeostasis and intracellular signaling, cell adhesion, gene expression, and glycoprotein folding. Several researchers have observed that CRT overexpression increased Ca²⁺ fluxes across endoplasmic reticulum (ER) but decreased mitochondrial Ca²⁺ and membrane potential. Thus, increased Ca²⁺ turnover between the two organelles might damage mitochondria, accounting for the increased susceptibility of cancer cells to apoptotic stimuli. Several studies have demonstrated that CRT-overexpressing cancer cells showed increased sensitivity to drug- or radiation-induced apoptosis, whereas CRT-deficient cells were more resistant to UV-induced apoptosis. Moreover, the overexpression of CRT can modulate radiation-induced apoptosis by suppressing Akt signaling and regulating p53 function via altered Ca²⁺ homeostasis.Although several studies have confirmed that GRP78 and CRT expressed in a wide variety of malignant tumors and correlated to tumor behavior, the investigation on the expression of GRP78 and CRT in patients with osteosarcoma, and the relation between tumor behavior and GRP78 or CRT, have not been carried out. In this work, we make atteept to detect the expression of GRP78 and CRT in patients with osteosarcoma and analyze the expression difference in tumor tissue and normal tissue, pre- and post-chemotherapy, metastasis and non-metastasis. According to these results, we infer the relation between the expression of GRP78 or CRT with tumor growth, metatasis, and chemotherapeutics. PARTâ… Experimental study on the Expression of GRP78 inpatients with osteosarcomaObjectiveSeveral studies have shown that GRP78 was overexpressed in multiple tumors, and was positive correlative to the degree of malignancy and invasiveness. Futhermore, chemotherapy could reduce the expression of GRP78. However, there were no related investigation on the expression of GRP78 in human osteosarcoma tissues, and the effect of GRP78 on metastasis and chemotherapy. In this work, we make atteept to detect the expression of GRP78 in patients with osteosarcoma and analyze the expression difference in tumor tissue and normal tissue, pre- and post-chemotherapy, metastasis and non-metastasis. According to these results, we decide the relation between the expression of GRP78 with tumor growth, metatasis, and chemotherapeutics. Furthermore, we expect that our work could provide experimental and theoretical evidence for the treatment and prognosis evaluation of the patients with osteosarcoma.Method1 Immunohistological stainingThe specimens from 30 patients with osteosarcoma were collected as experimental group, in which 5 cases were non-metastasis, 25 cases were metastasis, and 20 cases were pre-chemotherapy, 10 cases were post-chemotherapy. Furthermore, 30 specimens from normal tissues were collected as control group.2 RT-PCRTotal RNA was extracted from crushed tissue which including experimental and control group, and then was undertaken to RT-PCR using RT-PCR kit. And the electrophoresis maps were transfered into the image analysis system to detect expression intensity.3 Western-BlotThe protein was extracted from crushed tissue including experimental and control group, and then was undertaken to Western-Blot. Film will be scanned into the image analysis system measured intensity of expression. And the films were transfered into the image analysis system to detect expression intensity.Results1 Immunohistological stainingGRP78 was mainly located in cell endoplasmic reticulum. And histiocyte that its endochylema was stained as green was considered as positive cell which expressed GRP78. The expression level of GRP78 in tumor tissue was higher than normal tissue around the tumor, metastasis group higher than non-metastasis group, pre-chemotherapy group higher than post-chemotherapy group. There were significant differences according to the analytic results between tumor and normal tissues, metastasis and non-metastasis, pre-chemotherapy and post-chemotherapy.2 RT-PCRGRP78 was expressed in all specimens. The expression level of GRP78 mRNA in tumor tissue was higher than normal tissue around the tumor, metastasis group higher than non-metastasis group, pre-chemotherapy group higher than post-chemotherapy group. There were significant differences according to the analytic results between tumor and normal tissues, metastasis and non-metastasis, pre-chemotherapy and post-chemotherapy.3 Western-BlotGRP78 was expressed in all specimens. The expression level of GRP78 in tumor tissue was higher than normal tissue around the tumor, metastasis group higher than non-metastasis group, pre-chemotherapy group higher than post-chemotherapy group. There were significant differences according to the analytic results between tumor and normal tissues, metastasis and non-metastasis, pre-chemotherapy and post-chemotherapy. PARTâ…¡Experimental study on the Expression of Calreticulin inpatients with OsteosarcomaObjectiveSeveral studies have shown that CRT was expressed in multiple tumors, and was correlative to tumor growth and response to therapy. However, there were no related investigation on the expression of CRT in human osteosarcoma tissues, and the relationship among CRT and tumor behavior, chemotherapy. In this work, we make atteept to detect the expression of CRT in patients with osteosarcoma and analyze the expression difference in tumor tissue and normal tissue, pre- and post-chemotherapy, metastasis and non-metastasis. According to these results, we concluded the relation between the expression of CRT with tumor growth, metatasis, and chemotherapeutics. Furthermore, we expect that our work could provide experimental and theoretical evidence for the treatment and prognosis evaluation of the patients with osteosarcoma.Method1 Immunohistological stainingThe specimens from 30 patients with osteosarcoma were collected as experimental group, in which 5 cases were non-metastasis, 25 cases were metastasis, and 20 cases were pre-chemotherapy, 10 cases were post-chemotherapy. Furthermore, 30 specimens from normal tissues were collected as control group.2 RT-PCRTotal RNA was extracted from crushed tissue which including experimental and control group, and then was undertaken to RT-PCR using RT-PCR kit. And the electrophoresis maps were transfered into the image analysis system to detect expression intensity.3 Western-BlotThe protein was extracted from crushed tissue including experimental and control group, and then was undertaken to Western-Blot. Film will be scanned into the image analysis system measured intensity of expression. And the films were transfered into the image analysis system to detect expression intensity.Results1 Immunohistological stainingCRT was mainly located in intracavity of cell endoplasmic reticulum. And histiocyte that its endochylema was stained as green was considered as positive cell which expressed CRT. The expression level of CRT in normal tissue was higher than tumor tissue, non-metastasis group higher than metastasis group, post-chemotherapy group higher than pre-chemotherapy group. There were significant differences according to the analytic results between tumor and normal tissues, metastasis and non-metastasis, pre-chemotherapy and post-chemotherapy.4 RT-PCRCRT was expressed in almost all specimens. The expression level of CRT mRNA in normal tissue was higher than tumor tissue, non-metastasis group higher than metastasis group, post-chemotherapy group higher than pre-chemotherapy group. There were significant differences according to the analytic results between tumor and normal tissues, metastasis and non-metastasis, pre-chemotherapy and post-chemotherapy.5 Western-BlotGRP78 was expressed in almost all specimens. The expression level of CRT in normal tissue was higher than tumor tissue, non-metastasis group higher than metastasis group, post-chemotherapy group higher than pre-chemotherapy group. There were significant differences according to the analytic results between tumor and normal tissues, metastasis and non-metastasis, pre-chemotherapy and post-chemotherapy.ConclusionIn this present work, we first detected the expression of GRP78 and CRT in patients with osteosarcoma. Furthermore, we make attempt to analyze the expression difference in tumor tissue and normal tissue, pre- and post-chemotherapy, metastasis and non-metastasis. Using Immunohistological staining, RT-PCR and Western-Blot, we found that the expression level of GRP78 in tumor tissue was higher than normal tissue around the tumor, metastasis group higher than non-metastasis group, pre-chemotherapy group higher than post-chemotherapy group. However, the expression level of CRT in normal tissue was higher than tumor tissue, non-metastasis group higher than metastasis group, post-chemotherapy group higher than pre-chemotherapy group.In summary, the results of the present study indicate that GRP78 could promote tumor proliferation, survival and metastasis, and could make response to chemotherapy. Moreover, the overexpression of CRT could increase the sensitive of the patients with osteosarcoma to chemotherapy. Therefore, GRP78 and CRT could be applied as molecular target to analyze tumor behavior and therapeutic reaction. And in clinical work, GRP78 and CRT may represent novel biomarkers which could be used to determine the development and prognosis of osteosarcoma. Combination therapy suppressing GRP78 expression or promoting CRT expression could inhibit tumor growth, increase the sensitive to chemotherapy, suppress metastasis and improve prognosis, which may be applied as a new gene therapy for osteosarcoma. As a result, the use of GRP78 or CRT as a molecule target or medium of gene therapy for osteosarcoma would have a broad implemented prospect.