Clinical practice for years has proven that Salvia miltiorrhiza has anti-cancer potential and its application in the treatment of a variety of cancers has achieved surprising effects [13, 14]. However, most of the studies about Sal B, one of the major biologically active components of Salvia miltiorrhiza, were focused on its effects on cardiovascular disorders. There are few data available about the effect of this component on cancer. It was for this reason that Sal B was evaluated in our study for its chemopreventive and chemotherapeutic potential against OSCC.
In the present study we investigated whether Sal B has any inhibitory properties on OSCC cells and potentially malignant Leuk1 cells, in order to provide more documentation on the possible application of Sal B on OSCCs and oral premalignant lesions. We have shown that Sal B potently inhibited cell growth and induced apoptosis in OSCC cell lines CAL27 and SCC4. However, Sal B was much less active in Leuk1. The mechanisms accounting for this selective growth inhibition need to be further investigated. It needs to be noted that Sal B undergoes degradation in normal saline solution which will affect its anti-cancer effect. But we found in this study that Sal B still significantly inhibited the growth of CAL27 and SCC4 cells after treatment over 24 hours. We suppose its anti-cancer effect will be more potent if Sal B could be used in a solid state.
Results of another study from this laboratory showed that the growth inhibitory effect of Sal B was related to the apoptosis-inducing effect of Sal B (determined by flow cytometry analysis, data not shown). The detailed mechanism(s) of the pro-apoptotic effect of Sal B on OSCC cells remains unclear, as we have not attempted to investigate this aspect of action in this study. But this finding, together with our previous in vivo results, suggests that Sal B may be a good candidate for therapy in patients with these malignancies.
Further microarray-based expression profiling and quantitative RT-PCR analyses were performed to confirm whether anti-angiogenesis is one of the possible mechanisms of Sal B-induced growth inhibition in CAL27 and SCC4 cells, the two OSCC cell lines sensitive to the growth inhibitory effects of Sal B. The results showed that HIF-1α is down-regulated by ≥3-fold in both of the Sal B treated CAL27 cells and SCC4 cells. This result was consistent with our previous immunostaining studies. In our previous study, we observed that the formation of microvessels, as well as the expression of pro-angiogenic factors HIF-1α and VEGF, was inhibited in dysplasia and SCC by Sal B . HIF-1α is a transcription factor activated in response to cellular hypoxia. Being stabilized under decreased tissue oxygen concentration, it works as a cellular oxygen-sensing system, and trans-activates a large number of genes. Included among these are erythropoietin, glucose transporters, glycolytic pathway enzymes, and inducible nitric oxide synthase [[15–17]]. Discoveries have shown that hypoxia activates HIF-1α, which functions as master switches to induce expression of several angiogenic factors including VEGF, nitric oxide synthase (NOS), platelet-derived growth factor (PDGF) and Ang2. Alteration and over-expression of HIF-1α has been detected in a variety of solid tumors, including breast, lung, ovarian and oral cancer [18, 19]. These observations, together with our results, strongly implied that inhibition of HIF-1α activation by Sal B, which resulted in lowered expression of downstream pro-angiogenic genes, may be a key mechanism of cell growth inhibition and anti-angiogenesis on oral cancers. Their relative protein expression levels and whether such an action can be demonstrated in vivo remains to be confirmed.
However, the anti-angiogenic effect of Sal B seemed contradictive to some previous studies which showed that Sal B might improve microcirculation by augmenting VEGF expression and promoting angiogenesis. [[9–11, 20]]. Thrombospondins (TSPs) are known to inhibit neovascularization by induction of endothelial cell apoptosis through interaction with CD36 , inhibition of metalloproteinase activity , and inhibition of cell-cycle progression . In addition to these well-known effects on endothelial cell proliferation and apoptosis, maintenance of vascular integrity is another notable function of TSPs [[24–26]]. In this study, thrombospondin-2 (THBS2) expression was up-regulated by ≥3-fold in both of the Sal B treated CAL27 cells and SCC4 cells. Another study in our lab showed that in the Sal B treated samples, the mural cell coverage index was significantly higher than that of the control. And the organization of mural cells in the two groups of samples was dramatically different (data not shown). This suggests that Sal B may prevent the formation of new vessels by promoting vascular maturation. It is possible that the pro-angiogenic effect of Sal B induces formation of mature vessels with efficient irrigation function. The new vessels are different from the angiogenesis in tumors which leads to the formation of a poorly organized vasculature characterized by tortuous and leaky vessels unable to support efficient blood flow (Further investigation will carried out to clarify this point). The potent effect of Sal B on blood circulation may reduce the hypoxia stress in the local tissues, thus inhibit the uncontrolled formation of leaky vasculature. Based upon this information, the current results may be in line with those of previous studies.
Several angiogenesis-associated genes, including Tenascin-C, Osteopontin, TGF-β1, Cox-2, HGF, MMP-2 and MMP-9 also displayed variable expression in this study. Expression of these genes was changed ≥3-fold in CAL27 cells or SCC4 cells. This result was consistent with earlier studies which reported that Sal B attenuates LPS-induced Cox-2, MMP-2 and MMP-9 expression in human aortic smooth muscle cells. [27, 28] We found that these genes also play important roles in other ways that can affect cell signaling, the apoptotic pathway, cell metastasis, and other cellular behaviors. For example, expression of COX-2 was inhibited in Sal B treated OSCC cells. Over-expression of COX-2 is thought to contribute to carcinogenesis by stimulating cell proliferation , inhibiting apoptosis , and enhancing angiogenesis . Such genes as Tenascin-C, Osteopontin and MMP, which were reported to contribute to tumor metastasis [[32–35]], were inhibited by Sal B in this study. Therefore, this suggested that Sal B may exert multiple effects on oral carcinogenesis. Further research is required to investigate whether other mechanisms, as anti-metastasis, anti-oxidant and anticoagulation effect, will contribute towards the chemopreventive effect of Sal B on OSCC cells.