To date, despite cancer treatment has made great advancement and innovation based on the development of basic science, genetic engineering technology and big data analysis technology, cancer remains one of the most refractory diseases nowadays, and the number of patients has increased year by year, causing a heavy burden on families and society (2, 25, 26). Moreover, the frequently occurrence of multidrug resistant pathogenic microorganisms is another challenge for researchers (40). Therefore, the discovery and development of safer and novel drugs based on natural products has become one of the research hotspots (27–29). While as an emerging source of novel natural products, the endophytic actinomycete has attracted increasing attention to academics (30–32). Our study obtained an endophytic isolate, designated as LRE541 from the root tissues of Lilium davidii var. unicolor Cotton, and found that the secondary metabolites of LRE541 possess potent antitumor and antimicrobial potential by inhibiting a variety of malignancies and pathogens growth.
The isolate LRE541, well-characterized by the comparative analysis of 16S rDNA sequence, was assigned to Streptomyces sp. The phylogenetic relationship demonstrated that isolate LRE541 formed a distinct branch with the highest 16S rDNA sequence similarity of 98.81% to the type strain Streptomyces tauricus JCM4837T. Phenotypically, isolate LRE541 grew well on all the tested media with diverse aerial and substrate myceliums, but produced red diffusible pigment only on Gao's No. 1 medium. The extracellular enzyme tests found that LRE541 had the potential to yield various enzymes such as protease, amylase and lipase, which are industrially important. In addition, LRE541 could tolerate high pH value up to 12.0, a salinity of 6% NaCl, and temperature up to 37 °C. In sum, these physiological traits are consistent with the characteristics of streptomycetes that they are prolific and possess high adaptive capability for surviving in many unique niches (33–35), what′s more, reflecting the physiological flexibility of Streptomyces isolate to adverse environmental conditions (36, 37). Furthermore, the availability of a broad spectrum of carbon and nitrogen sources plays a vital role in the production of diverse secondary metabolites by Streptomyces sp. (38). Here, isolate LRE541 also exhibited the capability to utilize a wide range of carbon and nitrogen sources. This data provided an overview of the metabolite profile of LRE541, potentially serving as references for future research concerning fermentation optimization for higher yield of the desirable bioactive metabolites.
In view of the remarkable physiological capabilities above mentioned, the secondary metabolites (EtOAc extract) of isolate LRE541 was examined against nine representative human malignant tumors in vitro. The result revealed that the EtOAc extract showed cytotoxic activity towards all of the tumor cells with IC50 < 20 µg/mL, which is within the cut off point of cytotoxicity criteria recommended by the National Cancer Institute (NCI) for screening the cytotoxicity of crude plant extracts (39, 40). Moreover, the EtOAc extract exhibited cytotoxic activity against approximately 70% of the examined cancer cells with IC50 < 10 µg/mL, and marvelous antitumor potential against RKO, 7901 with IC50 value of only 0.021 and 0.29 µg/mL, respectively. However, it displayed a lower cytotoxic activity against human normal cell HPAEC with IC50 > 20 µg/mL. These results suggested that the EtOAc extract exerted potent and selective cytotoxic activity towards cancer cells. When tested against HepG2 cell line, the EtOAc extract (IC50 = 1.484 µg/mL) was shown to be more toxic than shikonin (IC50 = 4.3 µg/mL), a chemotherapeutic drug candidate, which was extracted from Lithospermum erythrorhizon Siebold & Zucc. (41, 42). When tested against MCF-7, several extracts from fungal endophytes (7 µg/mL ≤ IC50 ≤ 20 µg/mL) (43) and chromomycin SA (IC50 = 12.6 µg/mL) from Streptomyces sp. KML-2 inhabiting halophilic mines (44) showed less cytotoxic than the EtOAc extract (IC50 = 6.986 µg/mL). However, the EtOAc extract against Hela cell with IC50 value of 10.87 µg/mL, displayed less cytotoxic than Chromomycin SA (IC50 = 8.9 µg/mL) (44), while more cytotoxic than 6-gingerol and 6-shogaol extract (IC50 = 20.9 µg/mL) from Zingiber officinale Roscoe (45). The cytotoxic activity of the EtOAc extract against A549 (IC50 = 16.94 µg/mL) was found to be the weakest compared to the other cancer cells in this study, but examined to be more toxic than the extract from the endophyte Hypocrea lixii R18 (IC50 = 20.5 µg/mL) (46). Besides, the EtOAc extract from Streptomyces sp. LRE541 performed excellent antitumor effects towards K562 (IC50 = 8.106 µg/mL), SW1190 (IC50 = 12.98 µg/mL) and CAL − 27 (IC50 = 4.861 µg/mL), which represent leading causes of cancer-related death. Similarly, N. Kim et al. (47) reported the cytotoxic activities of salaceyins A and B from endophytic Streptomyces laceyi MS53 against SKBR3 with IC50 values of 3.0 and 5.5 µg/mL, respectively. Naphtomycin A from endophytic Streptomyces sp. CS showed cytotoxic activity against P388 and A549 cells with IC50 of 0.07 and 3.17 mM, respectively (48, 49). The cytotoxicity of 3-acetonylidene-7-prenylindolin-2-one, 7-isoprenylindole-3-carboxylic acid against A549 presented IC50 values of 3.3 and 5.1 µg/mL, respectively (50). Taken together, these results suggested that the endophytic isolate LRE541 colonizing the root tissues of Lilium davidii var. unicolor Cotton, could produce secondary metabolites with antitumor activities in vitro, but exhibit different anticancer potent against different cancer cells. This may be related to the specific molecular genetic background of different cancer cells, but further research will be needed.
It has been confirmed that apoptosis and necrosis are two patterns of cell death (51, 52). Compared to necrosis, an abnormal form of cell death, cell apoptosis regulated by various intra and extracellular signals and governed by several genes, plays a significant role in stress responses, control of normal cell proliferation and development of an organism (53, 54). While tumorigenesis is closely related to anti-apoptotic pathways (6). Thus, druginduced apoptosis of malignant cells is an efficient strategy in cancer therapy (6, 55). Our data presented that the EtOAc extract from the endophytic Streptomyces sp. LRE541 validly inhibited the cell viabilities of RKO and 7901 cells predominantly through the induction of apoptosis in a dose-dependant manner. Apparently, the apoptosis patterns between the two cells were greatly diverse, which suggested distinct mechanisms of the secondary metabolites actions occurring in the two cancer cells. Furthermore, previous studies have shown that cell cycle is likewise intimately associated with the tumorigenesis. Pathological or physiological apoptotic stimuli would greatly affect cell cycle progression, and disorder of cell cycle regulators is a common property of human cancer, which signifies that regulation of cell cycle progression in cancer cells is taken for an available method in the treatment of human malignancies (56, 57). This study, the EtOAc extract dramatically inhibited the cell proliferation of RKO and 7901 in a dose-dependent manner through inducing S phase arrest of cell cycle and apoptosis in vitro. Several studies had discovered that chemotherapeutic drugs originating from marine actinomycetes could cause cancer cell cycle arrest as well. Echinosporin and 7-deoxyechinosporin produced by Streptomyces albogriseolus A2002 were exhibited to block the cell cycle of HCT-15, K562 and tsFT210 cells mainly at the G0/G1 phase and promote apoptosis in these cells (58). Proximicins obtained from two rare actinomycetes Verrucosispora sp. MG-37 and Verrucosispora maris AB-18-032 isolated from marine sediments were found to block human gastric adenocarcinoma AGS cells at G0/G1 phase. Likewise, thiocoraline produced by the marine actinomycete Micromonospora sp. L-13-ACM2-092 caused an arrest in G1 phase of the cell cycle of human colon cancer cell lines (18). Collectively, chemotherapeutics with greater therapeutic efficiency and fewer side effects are of utmost desirability, and drug induced cancer cell death mode plays an important role in chemotherapy.
In the current study, the antagonistic activity of EtOAc extract associated with isolate LRE541 was screened in vitro condition by the agar well diffusion method against a panel of microbial pathogens. The results presented antimicrobial activities against Gram-positive, Gram-negative pathogens and yeast-like fugi with the concentration of EtOAc extract within 100 µg/mL. Interestingly, the antagonistic potent against the tested pathogens all displayed a dose-dependent manner, however, varied among different pathogens. This indicated diverse mechanisms of the EtOAc extract against those microorganisms. Similarly, the antagonism of endophytic Streptomyces sp. in this study has been discovered in other reports as well. For example, the endophytic Streptomyces sp. T3SB005 from root tissues of Thymus roseus exhibited antagonistic activities against three human pathogens (59); the culture filtrate of Streptomyces sp. HUST012 from Dracaena cochinchinensis Lour. was found against E. coli ATCC 25922 and MRSA ATCC 25923 with an inhibition zone of 18.9 mm (5). These findings suggested that endophytic streptomycetes may be exciting sources of bioactive compounds.
The antineoplastic and antimicrobial capacities of the EtOAc extract from isolate LRE541 suggest that the presence of antitumor and antimicrobial agents in the mixture of the extract may account for it. And we pursued this via ultra-high performance liquid chromatography-tandem mass spectrometry method. Based on the UHPLC-MS/MS analysis, the EtOAc extract was detected to contain thirty-nine compounds testified to exhibit various antitumor activities and cytotoxicity, besides, sixteen compounds proved to be antagonistic against diverse microorganisms. For example, ferulic acid, relative ratio of 15.3965% in the ESI (−) mode, as a new fibroblast growth factor receptor 1 (FGFR1) inhibitor, could inhibit the melanoma growth and angiogenesis using a melanoma model in vivo (60); similarly, 4-Hydroxybenzylalcohol, a promising anti-angiogenic agent, suppressed the vascularization and growth of newly developing CT26.WT tumors in vivo (61); while formononetin and catechin suppressed MCF-7 proliferation through blocking the cell cycle arrest or inducing apoptosis, respectively (62, 63); citral in combination with doxorubicin, and glycocholic acid with epirubicin, can dramatically increase the chemosensitivity of doxorubicin in human lymphoma Ramos cells, epirubicin in Caco-2 cells, respectively (64, 65). Sorbic acid and Lauric acid with relative ratios of 14.9% and 1.2% in the ESI (+)/(−) modes, respectively, have broad-spectrum antagonistic activities against most of the Gram-positive, Gram-negative pathogens and fungi (66, 67). Cefradine and Pleuromutilin are common broad-spectrum, high effective and low toxic antibiotics. Moreover, trans-Cinnamaldehyde and Methyl cinnamate due to their antimicrobial activities have been used as preservatives in the food industry (68, 69). As a whole, these chemical constitutes detected by UHPLC-MS/MS may be directly responsible for the antineoplastic and antimicrobial properties of the EtOAc extract.
It is reported that oxidative stress and chronic inflammation can develop into cancerous lesions (70, 71). Here, several natural phenolic compounds (rosmarinic acid, 4-Hydroxyphenylacetic acid and eugenol) and flavonoids compounds (eriodictyol, daidzein, glycitein) detected from the EtOAc extract, have been reported to possess excellent antioxidant activity (72–76). (-)-Caryophyllene oxide and cynaropicrin present in the extract had been demonstrated to have potent anti-inflammatory effect (77, 78). A NADPH oxidase inhibitor, 7-Methylguanosine (79), and cNIIIB nucleotidase inhibitor, apocynin (80), were all detected in the extract. Taken together, the above compounds may play an indirectly role in the antitumor activity of the EtOAc extract through antioxidant or anti-inflammatory effects, although further experimental evidence is necessary to testify their antineoplastic activity. Moreover, considering the potent antineoplastic activity of the EtOAc extract in the present study, the antitumor activity of the thirty compounds with relative ratio above 1% deserved to be further research.