| Nasopharyngeal carcinoma (NPC) is the most commonly seen epithelial malignancy in the nasopharynx, especially in Southern China. It usually manifests itself as a poorly differentiated carcinoma pathologically and holds a biological behavior being highly metastatic clinically. Because of its focus being very closed to the intracranial organs, it is very frequent to see such a tumor invading intracranially to cause intracranic metastasis in this way. It is still not very clear that the underlying mechanisms for such a pathological process, though many investigators have made a lot of work on this condition. Therefore, very important and worthful to practice is that to explore the mechanisms, especially at the molecular level, really associated with the pathogenesis and developing process of intracranial metastasis of NPC. Only in this way, can we prevent it from occurring and treat effectively once such a case occurring.Aiming at these goals, we have carried out a series of preliminary studies on the related mechanisms possibly underlying intracranial metastasis from NPC as follows.SECTION 1 Clinical observation on the intracranial metastasis potentiality among untreated NPC cases with different patternsMethods Sixty untreated NPC cases were included in this part of study and they were differentiated into different patterns based on a standardized syndrome differentiating system at first. Then, they were all determined the metastasis-relating indexes and followed up for one year in order to explore the relationship between the pattern and intracranial metastasis potentiality.Results The cases with the pattern of dual deficiency of Qi and Yin held significantly increased potentiality of intracranial metastasis than those with other two kinds of pattern within the following up period lasted for one year since the confirmation of NPC diagnosis (P<0.01).SECTION 2 Screening of the differentially expressed genes related with intracranial metastasis potentiality in the primary NPC tissues of nasopharynxMethods The differentially expressed genes related with intracranial metastasis potentiality in the primary NPC tissues were screened by use of XproTM HC-?plus cancer-relating gene chip of oligonucleotide (2748 cancer-relating genes). Theses procedures were carried out in a comparative way among various groups of tissue sample biopsied from the primary nasopharyngeal focus or the primary focus in the lung, i.e. the group of chronic nasopharyngitis, NPC group without metastasis, NPC group with lymph node metastasis in neck, NPC group with intracranial metastasis, NPC group with metastasis in both directions of neck and cerebrane and focus tissue samples of lung cancer with intracranial metastasis. Then, the differentially expressed genes relatively specific for intracranial metastasis were screened among these groups samples.Results Compared with those seen in other groups of tissue samples,257differentially expressed genes were screened out in the tissue samples of primary focus of NPC in nasopharyngnx with intracranial metastasis, with 101 genes expressing up regulated and 156 ones expressing down regulated.SECTION 3 Confirmation of intracranial metastasis-relating genes in the primary focus tissue of NPC or lung cancerMethods RT-PCR, immunohistochemist and Westernblot procedures were utilized to confirm the differentially expressed intracranial metastasis-relating genes in the primary focus tissue of NPC or lung cancer, which were determined through the analysis on gene expressing profile considering the candidates being associated relatively specific with intracranial metastasis of lesion among the all 6 groups of tissue samples.Results These kind of genes, like MMP-9, VEGF165 as two representative of these, were expressed in a closely related way with various metastasis potentiality of intracranial metastasis in the primary focus tissues. They could be ranked in an order from high to low according to the correlation with intracranial metastasis potentiality as in NPC group with metastasis in both directions of neck lymph node and intracranial way, in the group of lung cancer with intracranial metastasis, in NPC group with simple intracranial metastasis, in NPC group with neck node metastasis, in NPC group without metastasis and in the group of chronic nasopharyngitis.while the expressing level of nm23-Hl was in a reverse way. to that of VEGF165, MMP-9 in its activity expression among these cases with the same kind of pattern.SECTION 4 An analysis on the differential expressing characters of metastasis-relating genes in the primary focus tissue samples of untreated NPC cases with different patternsMethods Forty untreated NPC cases were firstly differentiated for their own patterns and then, a biopsy was made to take tissue samples from the nasopharynx of them. Expressing level determination of metastasis-relating genes nm23-H1 and VEGF165 was carried out among these samples to analyze the correlation of expressing activity of them with their firstly differentiated patterns.Results The expressing level of nm23-H1 was significantly lower in the tissue samples of primary focus in the nasopharynx among the untreated NPC cases with a pattern of dual deficiency of Qi and Yin than those with the other two kinds of pattern, while the expressing level of VEGF165 was in a reverse way to that of nm23-H1 in its activity expression among these cases with the same kind of pattern.SECTION 5 Ca2+ current features of sub-clonal cell lines of NPC with different metastatic potentiality Methods Sub-clonal NPC cell lines, with higher or lower metastatic potentiality respectively, were cultured and regenerated under the culturing conditions of 10% bovine serum,37?,5%CO2 and saturated humidity. Then, they were prepared as cell suspension respectively and cultured together with herbal medicinal serum prepared ealier. Following this, Ca2+ current features were determined respectively in a whole cell recording way from these cell lines with a different expressing level of nm23-H1 activity and different metastatic potentiality by use of the patch clamp techniques.The activity of cell moving was determined respectively in a wounding healing assay way.Results The main way of Ca2+ current in the sub-clonal cells with higher metastatic potentiality was the one induced by Ca2+influx started from Ca2+-releasing activated Ca2+current, with a very significant difference in the amplitude of Ca2+current with that of sub-clonal cells with lower metastatic potentiality (P<0.01). There was a significant difference in the activity of cell moving between the sub-clonal cell lines of NPC(P<0.01). Furthermore, the herbal medicine serum holds some intervening effects on the Ca2+current characters and cell moving activity of different sub-clonal NPC cell lines to suggest the potential application of herbal medicines in the prevention and treatment of intracranial metastasis from NPC focus.CONCLUSIONS Following conclusions could be made out based on the results derived from these studies.1. The untreated NPC cases with a pattern of dual deficiency of Qi and Yin held significantly higher potentiality of intracranial metastasis than those with either Qi and blood congealing or fire and toxin bonding.2. When compared with other clinical types of NPC, the cases with a type of intracranial metastasis showed relatively specific features of differential expression in tumor metastasis-relating genes in the tissues of primary focus in the nasopharynx, a part of them being expressed in an up regulated way and the other being expressed in a reverse way.3. In the tissue samples of primary nasopharyngeal focus among NPC cases with the type of intracranial metastasis, the fact of nm23-H1 gene being expressed in a down regulated way suggested that these cases hosted the tumor cells in the focus holding specular characters in their biological behavior and being responsible for their much elevated potentiality of intracranial metastasis.4. The activity of nm23-H1 was expressed in a significantly down regulated way while that of VEGF165 was expressed in a significantly up regulated way in the primary focus of NPC cases with a pattern of dual deficiency of Qi and Yin. This suggested that the factor of blood vessels might also be involved in the pathogenesis of intracranial metastasis from NPC, a developing tendency with much increased risk potentiality of intracranial metastasis. This condition might indicate a poorer prognosis for such cases.5. There was a significant difference in the amplitude of Ca2+ current and activity of cell moving between the sub-clonal cell lines of NPC, which expressed similar level of nm23-H1 activity but held different potentiality of metastasis, either higher or lower. This suggested that one of the solid difference at the cellular biological level might be the different mechanism of biological dynamics induced by Ca2+ current for these studied sub-clonal cell lines of NPC, with either higher or lower metastatic potentiality. Moreover, the intervening effects of herbal medicine on the Ca2+ current and activity of cell moving among different sub-clonal NPC cell lines may indicate that it is possible to use herbal medicines to block the pathogenesis process of intracranial metastasis from NPC focus in the nasopharynx. |
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