In Vitro and In Vivo Activity of Fomitopsis Pinicola (Sw. Ex Fr.) Karst Chloroform (Fpkc) Extract Against S180 Tumor Cells

Abstract

Background/Aims: Non-toxic fomitopsis is has been traditionally used in folk medicine in many countries for its anti-inflammatory and anti-vascular disease activities. The present study investigates the antitumor effect of Fomitopsis pinicola (Sw. Ex Fr.) Karst chloroform extract (FPKc) on S180 tumor cells in vitro and in vivo and we determined the underlying mechanisms. Methods: HPLC was employed to analyze the constituents of FPKc. In-vitro 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was performed to quantify the growth inhibition of FPKc; Propidium iodide (PI) exclusion assay and scanning electron microscopy (SEM) were used to observe the damage on the cell membrane and the changes of the cell morphology; Staining with Hoechst 33342/propidium iodide (HO/PI) and the application of the Annexin V-FITC/PI analysis permitted to observe the cell death triggered by FPKc; DNA damage and cell cycle arrest were detected by flow cytometry; Rhodamine 123 (RH123) and Cytochrome C were used as dyes to investigate the alterations of the mitochondria. In-vivo tumor inhibition and mice survival time were determined. Results: The results of the HPLC assay indicated that FPKc might contain DA (dehydroeburiconic acid), PA (pachymic acid), and ES (ergosterol), at percentages of 0.25%, 17.8%, and 10.5%, respectively. Concerning the study of the biological function, the results showed that FPKc exhibited preferential and significant suppression of proliferation on specific cell lines including S180, HL-60, U937, K562, SMMC-7721, and Eca-109 cells, which induced plasma membrane and cell morphology damages, triggering S180 tumor-cells late apoptosis and leading to DNA damage and S phase arrest. Mitochondria were suspected to play a vital role in these changes. In vivo data indicated that FPKc inhibited the solid tumor growth and prolonged the survival time of tumor-bearing mice. Moreover, FPKc provoked only little damage on normal cells in vitro and also on normal tissues in vivo. Conclusion: FPKc inhibited S180 tumor cells growth and prolonged the lifespan of mice. In vitro, we found that FPKc induced S180 tumor cells apoptosis and cell cycle arrest, possibly via the mitochondrial pathway.

Characterisation of Extracts and Anti-Cancer Activities of Fomitopsis pinicola

“…Cell cycle dysregulation is another hallmark of cancer [32] and may be a target of the mechanism of action of FPKc. This reasoning is supported by in vitro evidence showing the inhibition of cell proliferation; damage to cell membrane in sarcoma but not healthy cells; the triggering of S-phase cell cycle arrest; a decrease in MMP and release of mitochondrial cytochrome C [19]. Together, these in vitro studies show that F. pinicola extracts/compounds have anti-cancer activities which warrant further investigation.…”

“…In conclusion, further research is required to characterise the anti-cancer activities of F. pinicola as there is a paucity of data, particularly from in vivo and clinical studies. It would be useful to identify the bioactive components of F. pinicola and build on the research performed by Wang et al and Gao et al [19,26]. In particular, care must be taken to correctly identify each specimen using molecular techniques, prior to experimentation.…”

“…Underlying these hallmarks are mechanisms such as inflammation, genome instability and the creation of a tumour microenvironment [32]. Many of the in vitro studies outlined in Table 2 showed an increase in anti-oxidant activity [31], increase in apoptosis [19,26] or an upregulation of pro-apoptotic genes [21,22], and anti-inflammatory activity [23]. In addition, PARP, which is involved in DNA repair, genomic stability and programmed cell death, increased in response to treatment in a sarcoma cell line [20].…”

“…These studies are listed in Table 3. In two of the studies S-180 sarcoma cells were used to induce a xenograft [19,20], and in the remaining study, PC3 prostate cancer cells were used [13]. The extracts (ethanol and chloroform) were both active against the sarcoma xenograft and inhibited growth, but the powder obtained from an F. pinicola ethanol extract showed no activity against the prostate cancer xenograft.…”

“…The most obvious limitation of this review is the lack of certainty surrounding the identity of the specimens used in the studies we discuss. For example, Gao et al 2017 state that F. pinicola is traditionally categorized as Reishi [19], yet Reishi is identified as Ganoderma lucidum (Lingzhi) [28]. It is therefore unclear as to whether Gao et al studied G. lucidum or F. pinicola and the method of species identification is not stated.…”