The main finding of the present study is the in vitro anticancer efficacy of a novel extract obtained from Calendula Officinalis against various cancer cell lines derived from human or murine solid tumors of different etiologies. In some cases, this extract, LACE, achieved 100% inhibition of growth (Fig. 2, Table 1). Importantly, this inhibition effect is exerted on tumor cells from different solid tumors and is not specific to a single tumor cell tissue. The in vitro growth inhibition of LACE extract was similar to that reported for Taxol in tumor cell lines , as shown in Figure 2.
The principal chemical components of the aqueous extracts of Calendula Officinalis are: polysaccharides, proteins, fatty acids, carotenoids, flavonoids, triterpenoids and saponins. The carotenoids components may be excited by visible radiation. Laser treatment of the calendula extract is necessary to detect this biological activity. A similar calendula extract without laser treatment, CE, produced only a slight inhibition of tumor cell growth (Fig. 1b). The increase in this biological activity is due to treatment with laser radiation, which may induce conformational changes, excitation or degradation of different molecules of the CE extract.
Studies performed to assess the mechanisms involved in the above in vitro effects showed that LACE induced cell cycle arrest in G0/G1 (Fig. 3 and Table 2). Furthermore, mechanistic investigation showed that LACE-induced G1 arrest mainly mediated via a down-regulation of cyclins D1, D3, E y A, and CDK1-Cdc2, CDK2, CDK4 and CDK6 (Fig. 4). These novel data may have clinical relevance, since most human malignancies exhibit aberrations in cell cycle regulation . In fact, most anticancer agents derived from plants exert their effect via apoptosis induction in cancer cells [23–25]. The present data demonstrated that LACE induces apoptotic death and that the rate of apoptosis increases with higher LACE concentrations (Fig. 5, Table 3). The induction of apoptosis involved caspase-3-dependent mechanisms in some but not all tumor cell lines (Fig. 6), suggesting differential molecular determinants of apoptosis induction in different tumor cell lines.
In leukemia cell lines, LACE treatment inhibited 100% of growth in Jurkat cells and only 20% of growth in U937 cells, which may be explained by the different origin of the cells (lymphoma and monocytic, respectively) and/or the much higher growth rate of U937 cells, which double in number in less than 24 h. Interestingly, LACE induced proliferation of NKL leukemia cells (Fig. 2). The NKL cell line is dependent on IL-2 for its growth in vitro , indicating that it is in arrest phase of the cell cycle. Treatment with IL-2 or LACE produces cell cycle re-entry and similar proliferation values in NKL cells (Fig. 2). Likewise, a compound isolated from the fungus Coriolus Versicolor, Protein-bound polysaccharide K (PSK), also induces proliferation of NKL cells in absence of IL-2 . PSK has shown anticancer activity in vitro in experimental models and in human clinical trials [27, 28]. These anti-tumor activities can be largely attributed to activation of NK cells [29, 30].
The second novel finding of this study is that LACE treatment induces proliferation and activation of human PBLs (Fig. 1 and Fig. 7). The CE extract, without laser treatment, also produces a similar increase in PBL proliferation. PBLs, which do not proliferate in vitro and are found in G2/M arrest, re-entered cell cycle with LACE treatment (Fig. 3).
PBL subpopulations in LACE-induced proliferation were CD4+, CD19+, and mainly CD3+/CD16/56+ (Fig. 7). The latter correspond to NKT cells, which have been shown to recruit and promote a response by downstream effectors in an IFN-γ-dependent manner, activating both NK and CTL anti-tumor activity [31, 32]. These data, considered alongside the cytotoxic activity of LACE extract in tumor cell lines, indicate that it might have anticancer properties in vivo. In fact, preliminary results from our laboratory indicate that LACE can inhibit the growth of mouse tumor cells in vivo (unpublished results). Results obtained with PBLs and NKL cells indicate that cells in cell cycle arrest re-enter cell cycle with LACE treatment. In contrast, LACE treatment produces phase G1 cell cycle arrest in tumor cells in cell cycle (Fig. 3). Further research is required to determine whether a single active principle is responsible for this dual in vitro activity. Purification of LACE extract is in progress to identify its active principles. A further finding of this study was the low in vivo toxicity of this extract.
The LD50 for orally administered LACE was 550 mg/kg body weight in mice and 2750 mg/kg body weight in rats. Notably, the antiproliferative activity of LACE was not accompanied by systemic toxicity in mice or rats at a dose of 50 mg/kg body weight. The LACE extract showed in vivo anti-tumor activity in nude mice, the growth of Ando-2 tumor was reduced in a 60% (Fig 8). The anti-tumor efficiency of LACE was similar to obtained with other commonly used chemotherapy drug, paclitaxel. Furthermore, LACE produced higher prolongation of lifespan in tumor-bearing mice (Fig. 8).
The results of the present study are encouraging because LACE has shown significant inhibition of tumor growth in vitro and in vivo, therefore LACE or some components might be a promising chemotherapy candidate in treating cancers in clinic. Further experiments will focus on purification studies and the in vivo efficacy of LACE in other experimental mouse cancer models.