The role of pyrethroid derivatives in autophagy and apoptosis crosstalk signaling and potential risk for malignancies

Abstract

Pyrethroids and its derivatives widespread and uncontrolled continuous use has influenced multiple deleterious effects resulting in as a potential risk factor causing damage to the organ systems. Allethrin and prallethrin are extensively used yet their influences on human primary cells are very limited or under reported. The potential mechanisms by which allethrin and prallethrin modulates human primary cells, especially the molecular mechanisms or interconnectivity of autophagy-apoptosis, their clinical relevance in human subjects or patients are not well defined. In this current study, we've furnished the evidence that both allethrin and prallethrin user samples significantly induced Ccl2 mRNA expression, increased amount of reactive oxygen intermediate, inhibited membrane bound enzymes and altered membrane fluidity. Pyrethroid derivative users had induced levels of lipid peroxidation and induced binding activities of transcription factors(tfs) like CEBP-β and NF-AT. Pyrethroid derivatives induced autophagy, elicited intracellular Ca2+ concentration, calcineurin and regulated proapoptotic genes, DAPK1, Bim. Our current study presumably comprises the initial investigation of a very new mechanism of pyrethroid derivatives-moderated programed cell death in various cell sets or types, like human primary cells where-in this is a late event, is documented. Hence, current research-study might be significant in the various pyrethroid derivatives-allied hematological-related cancers and immunosuppressant or auto-immune disorders. In the foremost instance, we present data stating that pyrethroid derivatives induces multiple cell signaling cascades, like CEBP-β, NF-AT, ERK and MAPK having a role in autophagy thereby; synchronously effectively impact on the apoptosis, therefore causing hematological tumors and toxic or immune related disorders.

Keywords: Ccl2; allethrin; apoptosis; autophagy; prallethrin.

Figure 1. The effect of pyrethroid derivatives on CCL2 expression and ROS response.

(A) Effect of Allethrin and Prallethrin on Ccl2 expression and production in the allethrin and prallethrin derivative exposure users or treatments and healthy controls derived from human PBMCs (peripheral blood derived monocytes) and THP-1 cells: Cells were washed with PBS and allowed to rest 24 h. RNA was extracted and mRNA levels determined using quantitative real-time RT-PCR as described before (Raghavendra et al. 2013). (B) PBMCs were treated with Allethrin (100 μM), or Prallethrin (100 μM) for 24 h. Supernatants were analyzed for CCL2 levels using ELISA from Biosciences Inc, as described before (Raghavendra et al. 2013). (C) Human THP-1 cells were treated with Allethrin (100 μM), or Prallethrin (100 μM), DMSO (100 μM) and N-acetylcysteine, a well-known free radical scavenger - NAC (10μM) for 0, 1, 3, 6, 9, 12 and 24 h. ROS associated fluorescence levels were measured kinetically as described in the methods. ^* p < 0.05; ^** p < 0.01. N = 3–10.

Figure 2. The effect of pyrethroid derivatives on platelet membrane, lipid peroxidation and DNA binding.

(A) Effect of Allethrin and Prallethrin on platelet membrane. Two milliliters of samples in PBS was added to 2 μl of DPH solution (190 mM) in a cuvette and the fluorescent anisotropy was measured after incubation at 37°C for 20 min. The values denote mean ± S.D. independent determinations of N = 3. (B) Effect of Allethrin and Prallethrin derivative exposure users and healthy control on lipid peroxidation. Similarly, samples were used and lipid peroxidation was assayed by measuring malondialdehyde as represented. The values denote mean ± S.D. independent determinations of N = 3. (C) Docking interaction of Allethrin and Prallethrin with transcription factors CEBP-β (PDB id: 1GU4) and NFAT (PDB id: 2O93) was performed with the AutoDock4.0 program.

Figure 3. The effect of pyrethroid derivatives on induction of autophagy.

(A) RT- PCR analysis of autophagy genes in allethrin and prallethrin exposure users and healthy control PBMCs cells. Cells were washed with PBS and allowed to rest 24 h. RNA was extracted and mRNA levels determined using quantitative real-time RT-PCR as described before for Beclin-1 and LC3 genes, (Raghavendra et al. 2013). (B) PBMCs were treated with Allethrin (100 μM), or Prallethrin (100 μM), Doxorubicin (100 μM) and DMSO (100 μM) for 0, 6, 12 and 24 h in triplicates. After treatments, cells were fixed with paraformaldehyde (4%), washed three times with PBS, stained with MDC (50 μM) for 15 min, and washed three more times with PBS. Cells were collected, and fluorescence was measured and indicated as–fold, considering the unstimulated cell value as 1-fold from three independent experiments. (C) Cells were stimulated with Allethrin, Prallethrin, and Doxorubicin for 24 h and stained with Monodansylcadaverine (MDC) staining. The fluorescence of stained cells was visualized under a fluorescence microscope (scale bar, 10 μM). Error bars represent mean ± S.E. (Student’s t test). ^* p < 0.05; ^** p < 0.01; ^*** p < 0.001.

Figure 4. Pyrethroid derivatives elicited intracellular Ca2+ concentration, calcineurin and regulated proapoptotic genes, DAPK1/Bim.

(A–C) Fura-2/AM-loaded cells were resuspended either in Ca2+-containing buffer or Ca2+-free buffer containing EGTA. The ratio of fura-2 fluorescence intensity at the two excitation wavelengths (340/380 ratio) was monitored spectrophotometrically in a stirring cuvette during exposure to different concentrations ranging from 1μM - 100 μM Allethrin, Prallethrin. Results are representative single traces out of four experiments performed in duplicates. (D) THP-1 cells were treated with Allethrin (100 μM), or Prallethrin (100 μM) for 24 hr and calcineurin activity was assayed from cell extracts. Cells extract from Allethrin (100 μM), or Prallethrin (100 μM)-treated (24 h) cells was incubated with Cyclosporin A (2.5 μM) and calcineurin activity was assayed. (E) RT- PCR analysis of cell death genes in allethrin and prallethrin exposure users and healthy control PBMCs cells. Cells were washed with PBS and allowed to rest 24 h. RNA was extracted and mRNA levels determined using the quantitative real-time RT-PCR as described before for DAPK1 (Calcium/calmodulin–regulated (CaM-regulated) and Bim (Bcl-2-interacting modulator of cell death) genes.

Figure 5. Effect of pyrethroid derivatives on autophagy mediated signaling and impact of different inhibitors on Pyrethroid derivatives-mediated autophagy.

(A) and (a, a-1) Western blots were performed as described above. For this, THP1 cells were treated with Allethrin (100 μM) for the indicated time points. Cell lysates were then subjected to Western blot analysis for the indicated proteins. Blots were scanned and quantified using Image J. pAkt, pERK1/2, Beclin 1, LC3, p62 were normalized for loading using Akt, ERK2 or Tubulin. (B). Cells were pretreated with U01206 (10 μM for 1 h) and bafilomycin A1 (BafA1, 10 nM for 1 h), followed by stimulation with Allethrin (100 μM) or Prallethrin (100 μM) for 90 min and 24 h.phospho-ERK1/2 and Beclin 1 were determined by Western blot analysis. Representative blots are shown in the left column. N = 3. ^** p < 0.01; ^*** p < 0.001; ^**** p < 0.0001.

Figure 6. Effect of pyrethroid derivatives on caspase activity and cell death.

(A). Caspase activity analysis in allethrin and prallethrin exposure users and healthy control PBMCs cells. Cells were washed with PBS and allowed to rest 24 h. Caspase 3, 8, and 9 were detected from whole cell extracts using colorimetric substrate. The absorbance was taken at 405 nm and results represented in fold activation of caspases. (B). Effect of Pyrethroid derivatives in different human primary cells: Isolated neutrophils, epithelial cells and differentiated macrophages were incubated without or with Allethrin (100 μM), or Prallethrin (100 μM) for 24 h. Cytotoxicity was assayed and mean absorbance of triplicate samples were calculated and indicated as inhibition of cell viability considering untreated cells as 100% cell viability. (C). Nuclear fragmentation was detected from Allethrin (100 μM), or Prallethrin (100 μM) and Doxorubicin for 24 hr treated THP-1 cells by propidium iodide staining. The fluorescence of stained cells was visualized under a fluorescence microscope (scale bar, 15 μM). (D). THP-1 cells and PBMCs were incubated without or with Allethrin (100 μM), or Prallethrin (100 μM) for 24 h and assayed for cell viability using MTT dye. The results represented as inhibition of cell viability.