| Objective: To choose the effective concentration, it is nessary to compare with the effect of different concentrations of cardiac muscle cell culture medium(CMCM) on the proliferation of human nasopharyngeal carcinoma cells CNE-2 in vitro. Then the experiment will continue to study that CMCM has an effect on the migration of human nasopharyngeal carcinoma cells CNE-2 in vitro, which provides the experimental basis for further study. Observating the effects of CMCM lyophilized powder on the growth of human nasopharyngeal carcinoma(CNE-2) implanted in nude mice by oral administration and analyzing the effect of CMCM lyophilized powder on the body weight of nude mice, which provide the scientific theory evidence for the clinical practice of oral administration of CMCM lyophilized powder.Methods: 1. CMCM was collected by primary cultures of newborn rat cardiomyocyte, centrifuged and ultrafiltered through an amicon membrane which is a molecular weight cut off of 10 KD. At the same time, the low molecular weight fraction(<10 k D) was filtered through 0.22 μm filter, lyophilized by a vacuum freezing dryer and kept at-20℃ till use. 2. The abilities of different concentrations of CMCM on the cell proliferation of CNE-2 cells were tested by MTT assay. In addition, the abilities of effectiveconcentrations of CMCM on the cell migration of CNE-2 cells were observed by wound healing assay in vitro. 3. The models of the human nasopharyngeal carcinoma(CNE-2) xenograft transplanted in BALB/c-nude mice were established by cell suspension inoculation assay. Nude mice inoculated with the CNE-2 cells were randomly divided into 7 groups and began to give drugs when the tumor volume reached about 100mm3. The first group was treated with normal saline(25ml/kg/d,NS) through intraperitoneal injection and served as a control group. The second group was treated with CMCM lyophilized powder(15mg/kg/d) through intraperitoneal injection,which was solubed in NS(25ml/kg/d). The third group was treated with cisplatin(2mg/kg/d, DDP) through intraperitoneal injection, which was solubed in NS(25ml/kg/d) and served as a positive group. The fourth group was treated with normal saline(25ml/kg/d,NS) through oral administration and served as a control group. The fifth group was treated with low dose CMCM lyophilized powder(30mg/kg/d) through oral administration,which was solubed in NS(25ml/kg/d). The sixth group was treated with middle dose CMCM lyophilized powder(60mg/kg/d) through oral administration, which was solubed in NS(25ml/kg/d). The seventh group was treated with high dose CMCM lyophilized powder(120mg/kg/d) through oral administration, which was solubed in NS(25ml/kg/d). The drugs were administered for 14 consecutive days. The mental state,stool and eating of the nude mice were observed closely. The body weight of the nude mice and the tumor volume weremeasured by every three day. One day after the last treatment, the nude mice were sacrificed. Immediately, the tumors were removed and the tumor weight weren measured. The expression of cell apoptosis was observed by terminal deoxynucleotidyl transferase mediated d UTP nick end labeling(TUNEL) assay. The expression of cell proliferation was detected Ki67 by immunohistochemical assay. Results: 1. MTT assay data showed that compared with the control group of CNE-2 cell, the cell survival rates have a increasing effect on 1:4 dilution, 1:8 dilution and 1:16 dilution cardiac muscle cell culture medium(1/4 CMCM, 1/8 CMCM, 1/16 CMCM), but have a decreasing effect on 1:2 dilution and 1:1 dilution cardiac muscle cell culture medium(1/2 CMCM, 1/1 CMCM). 2. Wound healing assay results showed that untreated control group of CNE-2 cells showed complete wound healing with 24 h. In contrast, confluent monolayers of CNE-2 cells treated with CMCM or 1:2 dilution group showed clear wound width. There is no significant difference about the cell migration between the 1/2 CMCM and CMCM group, which showed CMCM can suppress the cell migration of CNE-2 cells in vivo at some extent concentration. 3. During the study,the tumor volume of each group represented a gradually increasing trend, but the speed of increasing is respectively decreased in experimental group compared to the control group. Compared with the control group, the tumor volume was shown significant differences by intraperitoneal injection from beginning the third day in the CMCM group(15mg/kg) and DDP group(2mg/kg). There aresignificant differences about the tumor volume treated with the low(30mg/kg), middle(60mg/kg) and high(120mg/kg) dose of CMCM lyophilized poweder by oral administration from beginning the third day. There are significant differences about the tumor volume between the abdominal group and the intragastric group at the same doge at the third, twelfth and fifteenth day. 4. In addition, the tumor weight were respectively delined in experimental group and there are significant differences about the tumor weight between the abdominal group and the intragastric group at the same doge, which suggested oral administration of CMCM lyophilized poweder can suppress the growth of human nasophyrngeal carcinoma(CNE-2) xenograft transplanted in nude mice. However, there is no significant difference about the inhibition rate of tumor between the abdominal group and the intragastric group at the same doge. 5. During the experimental period, the body weight of nude mice was increased in each group apart from the DDP group. There exists no significant difference on the body weight of nude mice by oral administration of CMCM lyophilized poweder, which suggested CMCM lyophilized poweder maybe an effective and safe drug for cancer therapy. 6. Immunohistochemical assay data showed that compared to the percentage of Ki67 positive cells of the control group(71.38±1.21%), the percentage of Ki67 positive cells treated with the CMCM group(15mg/kg) and DDP group were 48.86±2.13% and 25.55±3.2%, respectively. Compared to the percentage of Ki67 positive cells of the control group(75.70±7.17%),the percentage of Ki67 positive cells treated with low(30mg/kg), middle(60mg/kg) and high(120mg/kg) dose CMCM group were 60.65±4.23%, 51.04±4.58% and 41.42±8.24%, respectively. It is showed that oral administration of CMCM lyophilized poweder can inhibit the cell proliferation of human nasopharyngeal carcinoma cell CNE-2 in vivo. However, there are no significant differences about the percentage of Ki67 positive cells between the abdominal group and the intragastric group at the same doge. 7. TUNEL assay data showed that compared to the apoptotic rate of the control group(17.67±6.22%), the apoptotic rate of treated with the CMCM group(15mg/kg) and DDP group were 42.01±6.74% and 70.7±6.32%, respectively. Compared to the apoptotic rate of the control group(18.63±3.59%), the apoptotic rate of treated with low(30mg/kg), middle(60mg/kg) and high(120mg/kg) dose CMCM group were 30.23±6.02%, 51.63±11.09% and 60.61±8.71%, respectively. It is suggested that oral administration of CMCM lyophilized poweder can inhibit the cell apoptosis of human nasopharyngeal carcinoma cell CNE-2 in vivo. However, there are no significant differences about the apoptotic rate between the abdominal group and the intragastric group at the same doge. Conclusion: 1. Cardiac muscle cell culture medium may inhibit the cell proliferation and the cell migration of human nasopharyngeal carcinoma cell CNE-2 in vitro. 2. The cardiac muscle cell culture medium lyophilized poweder may be an effective and safe drug for cancer therapy and has a significant efficency on anticancer. 3. Oral administration of the cardiac muscle cell culture medium lyophilized poweder may inhibit the growth of human nasopharyngeal carcinoma cell CNE-2 implanted in nude mice. The mechanism of antitumor may be associated with inhibiting cancer cell proliferation and inducing tumor cell apoptosis. |
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