The inhibitors used were caspase 3 inhibitor caspase 8 inhibitor caspase 9 inhibitor general caspase inhibitor negative control and doxorubicin as a positive control ( as described in section 5. Kratom Hair Drug Test the positive control kratom legal in korea doxorubicin confirmed the assay works by showing a highly significant response for apoptosis. Thus this finding supported the kratom vendors that ship to indiana notion that there was no involvement of caspase executioner nor caspase initiator activation in cell death induced by high dose MSE. C o N ntr eg ol a (E M tive tO C M SE co H) a C sp. M E C .
Shaping genetic alterations in human cancer: The p53 mutation paradigm. Cancer Cell 12: 303-312. Future perspectives for the regulation of traditional herbal medicinal products in Europe. Phytomedicine 9: 572. Wild type p53 triggers a rapid senescence program in human tumor cells lacking functional p53.
M for MSE and MIT respectively (Chapter 2). The nature of cell death observed was unknown and to the best of my knowledge there are no reports or information kratom 7 hydroxymitragynine available on Mitragyna speciosa Korth toxicity on mammalian cells. In
this study therefore an attempt was made to characterise the MSE and MIT toxicity by looking at cell cycle distribution. Firstly attempt was made to look at the cell cycle distribution in different cell lines using flow cytometry approach. Propidium Iodide is one of the most common and recommended dyes to use to quantitatively assess DNA content by flow cytometry (Darzynkiewicz et al 2001).
The clonogenicity experiments using SH-SY5Y cells indicated that the chloroform contamination did not pose any obvious cytotoxic effects to level up of 500 uM concentrations which is far beyond that expectated to be in the MSE. M chloroform with MSE effects alone or chloroform alone (these data are from collaboration experiments with Thomas Randall ICL). Therefore it was assumed that the minor contamination of chloroform in both MSE and MIT was not contributing to the toxicity. We observed that MSE exerted dose dependent cytotoxicity with several human cancer cells both via trypan blue exclusion assay and clonogenicity assay.
SH-SY5Y cells and necrosis in HEK 293 cells. Cytological examination of SH-SY5Y cells after 48
hr treatment with MSE (24 hr treatment and 24 hr doubling time). Each photo is representative of 3 similar experiments with the same treatment concentration stained with WrightGiemsa staining. Magnification (x 1000). Cytological examination of HEK-293 cells after 48 hr treatment with MSE (24 hr treatment and 24 hr doubling time). Each photo is representative of 3 similar experiment with the same treatment concentration stained with WrightGiemsa staining.
Collectively kratom in high doses the current findings suggest that MSE induces a cycle arrest that appears to be independent of p53 pathway. In contrast MIT appears to induce cell cycle arrest that is p53 dependant. M respectively accompanied the cell death of the cell.
Effect of MSE and MIT on p53 protein levels SH-SY5Y a neuroblastoma cell known to have wild type p53 (Moll et al 1995 1996) was examined by immunoblotting as described in section 4. Image J version 1. The effects of MSE on p53 expression levels were assessed. The p53 protein level was found to be decreased in a dose-dependant manner especially at lower concentrations of MSE treatment for 24 hr as shown in fig. Further experiments were carried out to determine the time course of the down regulation or loss of p53 (Fig. MSE and control groups implying that this cell line expresses p53 protein and the lost of p53 protein seen at high doses was due to treatment effects.
PNAS 72: 979-983. Wild-type p53 can induce p21 and apoptosis in neuroblastoma cells but the DNA damage-induced G1 checkpoint function is attenuated. Clinical Cancer Research 5: 4199-4207. The potential for the use of cell proliferation and oncogene expression as intermediate markers during liver carcinogenesis.
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- This infers that MSE did not generate ROS which confirmed the earlier finding
- M of each inhibitor 30 minutes prior to adding the MSE
- The DNA profiles were determined using Modfit LT cell cycle analysis software (Verity Software Topsham ME)
- The Encyclopedia of Poisons and Antid
- B) appeared to be more resistant to the toxicity effects compared to SHSY5Y cells (Fig
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Animal models of neoplastic development. Biol (Basel) 106: 53-57. Facts and theories concerning the mechanisms of carcinogenesis. Laser capture microdissection microarrays and the precise definition of a cancer cell. H-mitragynine from Mitragyna speciosa in Thailand. Planta Medica 60: 580581. Mutational specificity of aflatoxin B1.
Thus it is suggested that apart from MIT there are other chemicals present in the leaves of Mitragyna specioa Korth contributing to the MSE cytotoxicity. A summary of the cytotoxic events leading to MSE or MIT induced SH-SY5Y cell death as discussed above are shown in fig. Mechanisms of MSE and MIT induced SH-SY5Y cells arrest and cell death. Arrows ( MSE; MIT) represent actual events occur in this study which leads to cell death. Dotted arrows ( MSE; MIT) represent possible mechanism of cell death as discussed in the text. The cell cycle arrest by MIT insult was associated with a positive link between p53 and p21; however cell cycle arrest due to MSE insult remains unclear due to loss of p53 and p21.