Low vitamin D status is considered as a risk factor for breast cancer and has prognostic significance. Furthermore, vitamin D deficiency increases after adjuvant cancer therapy, which alters bone metabolism increasing the risk of osteoporosis. It is now postulated that vitamin D supplementation in breast cancer treatment delays the recurrence of cancer thereby extending survival. We evaluated the impact of calcitriol and its low-calcemic analogs, PRI-2191 and PRI-2205, on the tumor growth, angiogenesis, and metastasis of 4T1 mouse mammary gland cancer. Gene expression analysis related to cancer invasion/metastasis, real-time PCR, ELISA, western blotting, and histochemical studies were performed. In vitro studies were conducted to compare the effects of calcitriol and its analogs on 4T1 and 67NR cell proliferation and expression of selected proteins. Calcitriol and its analogs increased lung metastasis without influencing the growth of primary tumor. The levels of plasma 17β-estradiol and transforming growth factor β (TGFβ) were found to be elevated after treatment. Moreover, the results showed that tumor blood perfusion improved and osteopontin (OPN) levels increased, whereas vascular endothelial growth factor (VEGF) and TGFβ levels decreased in tumors from treated mice. All the studied treatments resulted in increased collagen content in the tumor tissue in the early step of tumor progression, and calcitriol caused an increase in collagen content in lung tissue. In addition, in vitro proliferation of 4T1 tumor cells was not found to be affected by calcitriol or its analogs in contrast to non-metastatic 67NR cells. Calcitriol and its analogs enhanced the metastatic potential of 4T1 mouse mammary gland cancer by inducing the secretion of OPN probably via host cells. In addition, OPN tumor overexpression prevailed over the decreasing tumor TGFβ level and blood vessel normalization via tumor VEGF deprivation induced by calcitriol and its analogs. Moreover, the increased plasma TGFβ and 17β-estradiol levels contributed to the facilitation of metastatic process.

The discovery of tumor angiogenesis opened a new path in fighting cancer. The approval of different antiangiogenic agents, most targeting vascular endothelial growth factor (VEGF) signaling, has either increased the effectiveness of standard chemotherapy or even replaced it by offering better patient outcomes. However, an increasing number of preclinical and clinical observations have shown that the process of angiogenesis is far from clearly understood. Apart from targeting the VEGF pathway, novel strategies aim to influence other molecular factors that are involved in tumor angiogenesis. In addition, naturally occurring compounds seem to offer additional agents for influencing angiogenesis. The first concept of antiangiogenic therapy aimed to destroy tumor vessels, while it turned out that, paradoxically, antiangiogenic drugs normalized vasculature and as a result offered an improvement in chemotherapeutic delivery. In order to design an effective treatment schedule, methods for detecting the time window of normalization and biomarkers predicting patient response are needed. The initial idea that antiangiogenic therapy would be resistance-free failed to materialize and currently we still face the obstacle of resistance to antiangiogenic therapy.

A new series of betulin derivatives containing one or two pharmacophores bearing an acetylenic and carbonyl function at the C-3 and/or C-28 positions has been synthesized and characterized by 1H- and 13C-NMR, IR, MS and elemental analyses. The crystal structure of 28-O-propynoylbetulin was determined by X-ray structural analysis. All new compounds, as well as betulin, were tested in vitro for their antiproliferative activity against human SW707 colorectal, CCRF/CEM leukemia, T47D breast cancer, and against murine P388 leukemia and Balb3T3 normal fibroblasts cell lines. Most of the compounds showed better cytotoxicity than betulin and cisplatin used as reference agent. 28-O-Propynoylbetulin was the most potent derivative, being over 500 times more potent than betulin and about 100 times more cytotoxic than cisplatin against the human leukemia (CCRF/CEM) cell line, with an ID50 value of 0.02 μg/mL.