Fusaproliferin , a fungal phytotoxin shows rapid and potent cytotoxicity against pancreatic cancer cell lines

Nazia Hoque 1,3,4, Choudhury Mahmood Hasan 2, Md. Sohel Rana 3, Amrit Varsha5, Md. Hossain Sohrab 4*and Khondaker Miraz Rahman 5* 1 Department of Pharmacy, East West University, Dhaka, Bangladesh 2 Department of Pharmaceutical Chemistry, University of Dhaka, Dhaka-1000, Bangladesh 3 Department of Pharmacy, Jahangirnagar University, Savar, Dhaka, Bangladesh 4 Pharmaceutical Sciences Research Division (PSRD), BCSIR Laboratories, Dhaka,

In this study we investigated the fungal endophyte Fusarium solani, isolated from petiole of Aglaonema hookerianum Schott (Family:Araceae) growing in the forest of Sylhet and Chittagong Hill tracks of Bangladesh.It is a herb, stem erect, 40-50 cm or more tall, 1.5-2.0cm thick, internodes 1.5-3.0cm long.Leaves petiolate, petiole up to 24 cm long and grows in the shady areas of deep forest.A large number of tribal populations like Chakma, Marma, Murong, Tongchongya, Tripura, Chak, Khasia, Rheyang, Rakhain, Khumietc live as forest inhabitants in the remote areas throughout Sylhet and the Chittagong Hill Tracts, where there is no or poor modern medical systems of health care.The majority of them are dependent on the traditional system of treatment, which includes various indigenous medicinal plants of those areas [1]. A. hookerianum,locally known as Habinishak, is used by the mainstream traditional practitioners of Sylhet district for the treatment of hemorrhoids and arthritis.One tablespoon decoction prepared from the roots of A. hookerianum is taken orally twice daily for the treatment of gout.The sap from the root is taken for conjunctivitis and constipation by Chakma community.The leaf extract, applied to the whole body, is used for the treatment of hysteria by Tanchangya community [2]The petiole of A. hookerianum is used in the preparation of 'Shuktani', which is an ethno-medico recipe used in the treatment of stomach disorders like diarrhea, indigestion and dysentery by the Sylheti Bengali Community of Barak Valley, Southern Assam, India [3].Previous phytochemical study revealed the presence of alkaloid, glycoside, tannin, reducing sugar, saponin and gum in the ethanolic extract of A. hookerianum.The extract of this plant also showed potent antibacterial and cytotoxic activities [4].

Endophytic Fungus AHPE-4
For the identification of endophytic fungal isolates, slides prepared from cultures were stained with lactophenol cotton blue reagent and examined with a bright-field and phase contrast microscope. 6  Identification was based on morphological characteristics such as growth pattern, hyphae, the color of the colony and medium, surface texture, margin character, aerial mycelium, sporulation and production of acervuli, coloration of the medium, and the size and coloration of the conidia using standard identification manuals.The fungi were identified using relevant keys and taxonomic notes from various standard manuals [5].

Preparation of the plant and Initial Fungal Extracts
Total four endophytic fungi were isolated from different parts of A. hookerianum named AHPE-3, AHPE-4, AHLE-1 and AHLE-4.The fungus F. solani(AHPE-4), isolatedfrom the petiole of the plant A. hookerianumSchott after surface sterilization, was cultivated at 28 ± 2°C for 28 days on potato dextrose agar (PDA).The culture media were extracted with ethyl acetate for seven days in an air-tight flat bottom container with occasional shaking and stirring.This procedure was repeated for three times to obtain the crude extract.On theother hand, the powdered plant material (aerial part) of Aglaonema hookerianum wasextracted using a dichloromethane: methanol (1:1) solvent system.The crude extracts ofendophytic fungi, as well as the plant, were then filtered using sterilized cotton filter followed by Whatman no. 1 filter papers.The solvent was evaporated with a rotary evaporator at low temperature (40°C-50°C) and reduced pressure.
The dried crude extracts were stored at 4°C.The extracts of plant as wellas its associated endophytic fungal strains AHPE-3, AHPE-4, AHLE-1 and AHLE-4 were screened for probable cytotoxic activity using brine shrimp lethality bioass5.Brine Shrimp Lethality Bioassay Brine shrimp lethality bioassay was used [6] for evaluating cytotoxic activity using different concentrations of each extract using vincristine sulphate as the positive control.Theresult of the assay is shown in Figure S4.The ethyl acetate extract of Fusarium solani(AHPE-4) was found to be most active with an LC50 value of 17.05 μg/mL, whereas the positive control vincristine sulphate showed an LC50 value of 1.52μg/mL.

Figure S4 .
Figure S4.LC50 values of the endophytic fungi and the plant A. hookerianum in brine shrimp lethality bioassay.

Figure S5 .
Figure S5.MTT cell-viability assay profile in pancreatic (MIA PaCa2 and BXPC3) treated with gemcitabine and and breast (MDA MB 231 and MCF7) cancer cell lines treated with doxorubicinfor 24 h.