In this study, we examined the sequential expression of several matrix metalloproteinases (MMPs), tissue inhibitors of metalloproteinases (TIMPs), and growth factors as well as the presence of apoptosis in a model of pulmonary fibrosis induced in rats with paraquat and hyperoxia. Animals showing neither clinical nor morphological changes with this double aggression were classified as "resistant". Rats were killed at 1, 2, 3, and 6 wk, and lungs were used for collagen content, gene expression by real-time PCR, gelatinolytic activity by zymography, apoptosis by in situ DNA fragmentation, and protein localization by immunohistochemistry. Our results showed a significant decrease of collagenases MMP-8 and MMP-13, with an increase of TIMP-1 and transforming growth factor-beta. Immunoreactive TIMP-1 was increased in experimental rats and primarily localized in alveolar macrophages. Expression of gelatinases MMP-2 and MMP-9 mRNAs was not affected, but lung zymography revealed an increase in progelatinase B, progelatinase A, and its active form. Epithelial apoptosis was evident from the first week, whereas at later periods, interstitial cell apoptosis was also noticed. Resistant animals behave as controls. These findings suggest that an imbalance between collagenases and TIMPs, excessive gelatinolytic activity, and epithelial apoptosis participate in the fibrotic response in this experimental model.

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Voltage-gated sodium (NaV) channels have been related with cell migration and invasiveness in human cancers. We previously reported the contribution of NaV1.6 channels activity with the invasion capacity of cervical cancer (CeCa) positive to Human Papilloma Virus type 16 (HPV16), which accounts for 50% of all CeCa cases. Here, we show that NaV1.6 gene (SCN8A) overexpression is a general characteristic of CeCa, regardless of the HPV type. In contrast, no differences were observed in NaV1.6 channel expression between samples of non-cancerous and cervical intraepithelial neoplasia. Additionally, we found that CeCa cell lines, C33A, SiHa, CaSki and HeLa, express mainly the splice variant of SCN8A that lacks exon 18, shown to encode for an intracellularly localized NaV1.6 channel, whereas the full-length adult form was present in CeCa biopsies. Correlatively, patch-clamp experiments showed no evidence of whole-cell sodium currents (INa) in CeCa cell lines. Heterologous expression of full-length NaV1.6 isoform in C33A cells produced INa, which were sufficient to significantly increase invasion capacity and matrix metalloproteinase type 2 (MMP-2) activity. These data suggest that upregulation of NaV1.6 channel expression occurs when cervical epithelium have been transformed into cancer cells, and that NaV1.6-mediated invasiveness of CeCa cells involves MMP-2 activity. Thus, our findings support the notion about using NaV channels as therapeutic targets against cancer metastasis.

Serum samples from 38 healthy women and 55 women with different types of cervical lesions were investigated for the presence of antibodies to Ras and against E4 and E7 proteins of human papillomavirus type 16 (HPV-16). Our results showed that anti-E7 antibodies were closely associated with cervical cancer (75%), as previously reported. Interestingly, E4 antibodies showed higher prevalence in condyloma lesions (79%; 11/14) than in cervical cancer (60%; 12/20). We also identified 11% (4/38) of healthy individuals as positive for E4 antibodies, which suggests an early immune recognition of this protein. Patients with condyloma and cervical intraepithelial neoplasia (CIN) also showed higher prevalences of Ras antibodies (approximately 40%) than cervical cancer patients (10%; 2/20). By sequencing part of the ras genes and using different Ras antigens, we showed that serum antibodies from patients were not directed to a Ras mutation, since wild-type cHa-Ras protein was recognized by these antibodies. In addition, patients positive for Ras antibodies (94%) were also positive for E4 antibodies, suggesting an association between these. The high prevalence of antibodies against Ras and E4 proteins in pre-malignant lesions opens the possibility of using both antibodies as early markers for potential cervical cancer patients.