Inhibition of Human Gastric Carcinoma Cell Growth in Vitro and in Vivo by Cladosporol Isolated from the Paclitaxel-Producing Strain Alternaria alternata var. monosporus

Abstract

Paclitaxel (Taxol) was isolated from the bark of the Pacific yew tree Taxus brevifolia in 1971, 1) and has become instrumental in treating refractory ovarian and breast cancers. 2,3) However, because the yield of paclitaxel from bark is only approximately 0.01% (w/w), 4,5) and bark stripping destroys scarce plant materials, scientists are still searching for new compounds in the yew tree. Therefore microbial fermentation methods are used in the production of paclitaxel. We have obtained the high-yield paclitaxel-producing strain Alternaria alternata var. monosporus from the bark of a yew tree in Kunming, China. During the extraction of the hyphae, another compound cladosporol ( Fig. 1) was obtained in large amounts. Since both cladosporol and paclitaxel can be obtained from the same strain, we examined whether cladosporol also has antitumor effects. In this study, we report the preparation of cladosporol and its antitumor effects against various human cancer cell lines and in nude mice bearing MGC-803 gastric cancer xenografts. MATERIALS AND METHODS MaterialsThe microorganism strain A. alternate var. monosporus was obtained from the bark of a yew tree in Kunming, Yunnan province, China, and identified by Prof. Shijin Deng, Liaoning University, China. All reagents and chemicals used were of analytical grade and commercially available.Animals Female BALB/c nude mice, 35-40 d old and weighing 18-22 g, were supplied by Shanghai Slac Laboratory Animal Limited Company. The mice were raised in airconditioned rooms under controlled lighting (12-h light/dark) and fed with standard laboratory chow and water ad libitum.Cell Lines and Cell Culture The human hepatoma carcinoma cell line SMMC-7721, human breast carcinoma cell line Bcap-37, human renal carcinoma cell line 786-0, human leukemia cell line K562, human prostate carcinoma cell line PC-3, and human gastric carcinoma cell line MGC-803 were obtained from the Cell Bank of the Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences. Cells were cultured in RPMI 1640 medium (Gibco Invitrogen Co., Grand Island, NY, U.S.A.) supplemented with 10% FBS, 100 U/ml of penicillin, and streptomycin (Sigma, U.S.A.) at 37°C in a humidified 5% CO 2 -95% air incubator under standard conditions. 6) To maintain exponential growth, cells were seeded at 1ϫ10 5 cells/ml and passaged every 4 to 5 d.Colorimetric MTT Assay Cell growth inhibition was evaluated using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay in triplicate. 7)Cells were treated with different concentrations (4.8, 2.4, 1.2, 0.6, 0.3 mg/ml) of cladosporol for specified times at 37°C. Control cells grown only in culture media containing 0.1% methanol. After incubation for 48 h, absorbance (A) was measured at 570 nm. The survival ratio (%) was calculated using the following equation: survival ratio (%)ϭ(A treatment / A control )ϫ100. The IC 50 value was taken as the concentration that caused 50% inhibition of cell viability and calculated using the Logit method. Measurement ...

Cladosporol a stimulates G1‐phase arrest of the cell cycle by up‐regulation of p21waf1/cip1 expression in human colon carcinoma HT‐29 cells

“…Growth recovery was even more evident when cells were treated for 24 or 48 h with the drug (5 µM) and then left in culture for additional 96 h in the absence of the drug (Supplementary data1). These results are consistent with those reported by Chen et al [33] who showed no toxicity induced by cladosporol in gastric cancer cells xenografted into nude mice. Finally, cladosporol A‐treated cells detached from the plate surface with a typical rounded‐up morphology as observed with a light transmission microscope (Figure 2C).…”

“…Along this reasoning, Chen et al [33] have recently demonstrated that the growth of the human gastric carcinoma derived cells (MGC‐803) is inhibited by cladosporol isolated from the paclitaxel‐producing strain Alternaria alternata var: monosporus . Here, we tested the antiproliferative properties of cladosporol A on three CRC derived cell lines (HT‐29, SW480, and CaCo‐2) to elucidate the underlying molecular mechanisms.…”

Metabolites from Alternaria Fungi and Their Bioactivities

“…Several metabolites are unique to one Alternaria species, but most metabolites are produced by more than one species. Occurrences of the isolated metabolites from Alternaria fungi are listed in Table 1 [ 16 , 17 , 18 , 19 , 20 , 21 , 22 , 23 , 24 , 25 , 26 , 27 , 28 , 29 , 30 , 31 , 32 , 33 , 34 , 35 , 36 , 37 , 38 , 39 , 40 , 41 , 42 , 43 , 44 , 45 , 46 , 47 , 48 , 49 , 50 , 51 , 52 , 53 , 54 , 55 , 56 , 57 , 58 , 59 , 60 , 61 , 62 , 63 , 64 , 65 , 66 , 67 , …”

“…Typical examples included AAL toxins 3 – 10 from Fusarium species [ 5 , 136 , 137 ], helvolic acid ( 117 ) from Aspergillus species [ 138 ] and Pichia species [ 139 ], paclitaxel (taxol, 61 ) from yew trees ( Taxus spp.) [ 140 ], resveratrol ( 252 ) from a variety of plant species such as Vitis vinifera , Polygonum cuspidatum and Glycine max [ 141 ], besides these metabolites from Alternaria species [ 43 , 53 , 130 ].…”

Antitumor Potential of Seaweed Derived-Endophytic Fungi

“…Cladodosporol H (15) (Figure 1) exhibited significant cytotoxicity against A-549, Huh7 and LM3 cell lines with IC 50 values of 5.00, 1.00, and 4.10 µM, respectively, clodosporol C (16) (Figure 1) showed activity against H446 cell line with IC 50 value of 4.00 µM [51]. Cladosporols are known to be unique to the fungal genus Cladosporium and have attracted the attention of researchers because of their ability to stimulate cell cycle G1-phase arrest in human colon carcinoma HT-29 cells, thus demonstrating their promising antitumor activity [51,[70][71][72].…”