PRP4 Kinase Domain Loss Nullifies Drug Resistance and Epithelial-Mesenchymal Transition in Human Colorectal Carcinoma Cells

Ahmed, Muhammad Bilal,Islam, Salman Ul,Sonn, Jong Kyung,Lee, Young Sup Korean Society for Molecular and Cellular Biology 2020 Molecules and cells Vol.43 No.7

We have investigated the involvement of the pre-mRNA processing factor 4B (PRP4) kinase domain in mediating drug resistance. HCT116 cells were treated with curcumin, and apoptosis was assessed based on flow cytometry and the generation of reactive oxygen species (ROS). Cells were then transfected with PRP4 or pre-mRNA-processing-splicing factor 8 (PRP8), and drug resistance was analyzed both in vitro and in vivo. Furthermore, we deleted the kinase domain in PRP4 using Gateway™ technology. Curcumin induced cell death through the production of ROS and decreased the activation of survival signals, but PRP4 overexpression reversed the curcumin-induced oxidative stress and apoptosis. PRP8 failed to reverse the curcumin-induced apoptosis in the HCT116 colon cancer cell line. In xenograft mouse model experiments, curcumin effectively reduced tumour size whereas PRP4 conferred resistance to curcumin, which was evident from increasing tumour size, while PRP8 failed to regulate the curcumin action. PRP4 overexpression altered the morphology, rearranged the actin cytoskeleton, triggered epithelial-mesenchymal transition (EMT), and decreased the invasiveness of HCT116 cells. The loss of E-cadherin, a hallmark of EMT, was observed in HCT116 cells overexpressing PRP4. Moreover, we observed that the EMT-inducing potential of PRP4 was aborted after the deletion of its kinase domain. Collectively, our investigations suggest that the PRP4 kinase domain is responsible for promoting drug resistance to curcumin by inducing EMT. Further evaluation of PRP4-induced inhibition of cell death and PRP4 kinase domain interactions with various other proteins might lead to the development of novel approaches for overcoming drug resistance in patients with colon cancer.

PRP4 kinase induces actin rearrangement and epithelial-mesenchymal transition through modulation of the actin-binding protein cofilin

Islam, Salman Ul,Ahmed, Muhammad Bilal,Lee, Su Jin,Shehzad, Adeeb,Sonn, Jong Kyung,Kwon, Oh-Shin,Lee, Young Sup Elsevier 2018 Experimental cell research Vol.369 No.1

<P><B>Abstract</B></P> <P>Cell actin cytoskeleton is primarily modulated by Rho family proteins. RhoA regulates several downstream targets, including Rho-associated protein kinase (ROCK), LIM-Kinase (LIMK), and cofilin. Pre-mRNA processing factor 4B (PRP4) modulates the actin cytoskeleton of cancer cells via RhoA activity inhibition. In this study, we discovered that PRP4 over-expression in HCT116 colon cancer cells induces cofilin dephosphorylation by inhibiting the Rho-ROCK-LIMK-cofilin pathway. Two-dimensional gel electrophoresis, and matrix-assisted laser desorption/ionization time-of-flight mass-spectrometry (MALDI-TOF MS) analysis indicated increased expression of protein phosphatase 1A (PP1A) in PRP4-transfected HCT116 cells. The presence of PRP4 increased the expression of PP1A both at the mRNA and protein levels, which possibly activated cofilin through dephosphorylation and subsequently modulated the cell actin cytoskeleton. Furthermore, we found that PRP4 over-expression did not induce cofilin dephosphorylation in the presence of okadaic acid, a potent phosphatase inhibitor. Moreover, we discovered that PRP4 over-expression in HCT116 cells induced dephosphorylation of migration and invasion inhibitory protein (MIIP), and down-regulation of E-cadherin protein levels, which were further restored by the presence of okadaic acid. These findings indicate a possible molecular mechanism of PRP4-induced actin cytoskeleton remodeling and epithelial-mesenchymal transition, and make PRP4 an important target in colon cancer.</P> <P><B>Highlights</B></P> <P> <UL> <LI> PRP4 is involved in pre-mRNA splicing and cell signalling. </LI> <LI> PRP4 modulates the actin cytoskeleton of cancer cells via RhoA activity inhibition. </LI> <LI> PRP4 induces cofilin dephosphorylation by inhibiting the Rho-ROCK-LIMK-cofilin pathway in HCT116 cells. </LI> <LI> Dephosphorylation of cofilin results in F-actin stabilization, re-distribution of cytoplasmic actin, formation of actin stress fibers, and inhibition of cell motility. </LI> <LI> PRP4 over-expression induces the expressions of PP1A, which directly or indirectly dephosphorylates cofilin, resulting in actin cytoskeleton rearrangement, downregulation of E-cadherin, and EMT induction. Cofilin activation may be associated with EMT properties, and promotes the progression of human colon cancer. </LI> </UL> </P> <P><B>Graphical abstract</B></P> <P> <B>Proposed model for PRP4-induced cofilin and MIIP dephosphorylation and epithelial-mesenchymal transition (EMT) induction</B>. PRP4 over-expression results in cofilin and MIIP dephosphorylation, causing actin dynamics to increase, which may lead to EMT. Another proposed pathway for EMT induction by dephosphorylated MIIP is illustrated in the black-dotted panel. MIIP may inhibit the Rac1 signaling pathway through PAK1 (Rac1 downstream target) binding competition, which results in reduced lamellipodia formation and, finally, EMT.</P> <P>[DISPLAY OMISSION]</P>

범안저레이저광응고술 전 후 망막정맥충만시간 변화와 증식당뇨망막병증 진행과의 관계

장정운,두하영,양연식 대한시과학회 2009 대한시과학회지 Vol.11 No.2

목적: PRP(범안저레이저광응고술) 시행 전에 측정한 망막정맥충만시간의 절대치 뿐만 아니라, PRP 시행 전과 후에 그 변화량을 평가함으로써, 그 변화량과 신생혈관 진행과의 연관성을 규명하고자 하였다. 방법: 증식당뇨 망막병증으로 본원에서 PRP를 시행한 환자 중 PRP 전과 후에 형광안저촬영을 시행하였던 환자 32안을 주사형 레이저검안경을 이용한 형광안저촬영 시행 후 2주 내에 PRP를 시작하였고 PRP 시행 3~5개월 후에 다시 형광안저촬영을 시행하였다. 결과: 망막정맥충만 시간의 PRP 전 후의 변화는 안정화된 군에서는 -0.99±1.60초 감소하였고 불안정화된 군에서는 0.30±1.69초 증가하였으며 독립 T-검정상 망막정맥충만시간의 변화는 통계적으로 유의하였다(P Purpose: To measure venous filling time before the operation PRP(Panretinal photocoagulation), to evaluation Change of before and after PRP as well as relation of variation to PRP and progress of new blood vessels. Methods: We performed 32 eyes, who had PDR(proliferative diabetic retinopathy) during the operation of PRP patients, before and after PRP in video fluorescein angiogram using SLO(Scanning laser opthalmoscope). To started PRP within 2 weeks after SLO and the PRP to start again after 3-5 months of video fluorescein angiogram using SLO. Result: Change of venous filling time before and after the PRP decrease -0.99±1.60 second of regreseed group and increase 0.30±1.69 second of complicated group. In the T-test was significant(P

Characterization of the cytokine profile of platelet rich plasma (PRP) and PRP-induced cell proliferation and migration: Upregulation of matrix metalloproteinase-1 and -9 in HaCaT cells

Hong-Bum Park,양정희,Kwang-Hoe Chung 대한혈액학회 2011 Blood Research Vol.46 No.4

Background :The underlying rationale of platelet rich plasma (PRP) therapy is that an injection of concentrated PRP at the site of injury may promote tissue repair via cytokine release from platelets. The molecular mechanisms of PRP therapy in the skin wound healing process are not well understood at present, and would benefit from clarification. Methods :PRP was stimulated with angonists for 5 min, and cytokine profile analysis was performed. To investigate the wound healing activity of PRP, cell proliferation and migration analyses were performed in skin cells. The effects of PRP were analyzed on the expression and activity of matrix metalloproteinase (MMP)-1, -2, -9, and the activation of transcription factors. Results :Thrombin was found to be a strong stimulator of PRP activation to release growth factors and chemokines. PRP induced cell proliferation and migration in HUVECs, HaCaT cells, and HDFs, as well as MMP-1and MMP-9 expression in HaCaT cells, but PRP did not have a significant effect on the expression or activity of MMPs in HDFs. The transcription factors, including signal transducer and activator of transcription-3 (STAT-3) were found to be phosphorylated following PRP treatment in HaCaT cells. Conclusion :In this study, we have identified the cytokine profile of activated PRP after agonist stimulation. We have shown that PRP plays an active role in promoting the proliferation and migration of skin cells via the regulation of MMPs, and this may be applicable to the future development of PRP therapeutics to enhance skin wound healing.

Mycosporine-like amino acids (MAAs) 처리에 따른 배양세포 내 스크래피 프리온 단백질의 형성증가

이지현,모상현,류종석,김대환,Lee, Jihyun,Moh, Sang-Hyun,Ryou, Chongsuk,Kim, Dae-Hwan 한국미생물·생명공학회 2015 한국미생물·생명공학회지 Vol.43 No.2

Prion은 양의 scrapie, 소의 bovine spongiform encephalopathy와 사람의 CJD와 같은 다양한 신경 퇴행성 질환을 유발시키는 단백질 병원체이다. 정상 prion 단백질인 PrP^C가 병원성 PrP^Sc로 바뀌는 과정에 대해서는 많은 연구가 진행되었고, PrP^Sc로의 단백질 구조 변화가 다양한 환경적 요소에 의해서 영향 받는 것으로 추측된다. 바다조류로부터 분리된 MAAs는 다양한 스트레스 환경에서 조류를 보호해주는 것으로 알려져 있다. 이와 같은 사실에 기초하여 mycosporineglycine, porphyra-334와 shinorine 3종의 MAAs로 처리한 prion 감염 신경세포 주에서 prion 단백질 축적의 변화를 평가하였다. PK 저항성을 갖는 PrP^Sc를 western blot 방법으로 확인한 결과, MAA에 의해서 PrP^Sc 단백질의 증식을 관찰하였다. Prions are proteinaceous infectious particles that cause neurodegenerative diseases, such as scrapie in sheep, bovine spongiform encephalopathy in cattle and Creutzfeldt-Jakob disease (CJD) in humans. Although the detailed process, regarding the abnormal conversion of prion proteins (PrP), remains to be fully elucidated, a number of environmental factors appear to affect the formation of misfolded PrP, termed PrP^Sc. Because oceanic algae contain mycosporine-like amino acids (MAAs), which exhibit cellular defensive activities under a variety of stress conditions, we investigated the level of PrP^Sc in prion-infected neuroblastoma cells using mycosporine-glycine, porphyra-334 and shinorine. When judged by the level of protease-resistant PrP^Sc in western blots, porphyra-334 and shinorine increased the level of PrP^Sc in cells, but mycosporine-glycine did not. The current results indicate that the MAAs tested in this study enhance the formation of PrP^Sc.

가토두개골 골결손부에 혈소판풍부혈장(PRP)과 혈소판풍부피브린 (PRF)의 적용시 골형성인자 발현 연구

김영삼 ( Young Sam Kim ),이덕원 ( Deok Won Lee ),류동목 ( Dong Mok Ryu ),지유진 ( Yu Jin Jee ),최용석 ( Yong Suk Choi ) 한국조직공학·재생의학회 2009 조직공학과 재생의학 Vol.6 No.4

The aim of the present study is to evaluate the bone morphogenic factor of PRF and PRP on rabbit calvaria. Twelve healthy rabbits, weighing 2.5~3 kg, were used in this experiment. 8 mm diameter 4 hole were prepared on rabbit calvaria, each hole was filled with autologous bone, PRP and PRF. One defect site was filled with nothing as control group. According to experiment schedule, rabbits were sacrificed at 1, 2, and 6 weeks after surgery. Specimen were obtained using 10 mm trephin drill for real time RT-PCR and total calvaria were separated for H&E stain. RT-PCR was measured for mRNA expression of BMP-2, type I collagen, and osteopontin as a bone morphogenic factor. As a result, mRNA expression of BMP-2 in autologous bone group was higher than PRP and PRF group. PRP and PRF groups were higher than control group. In case of mRNA expression of Type I collagen, PRP and PRF groups progressively increased from 1 week to 6 weeks. In case of mRNA expression of OPN, PRF was higher at 2 weeks than 1 and 6 weeks. These result showed that PRP and PRF were more effective on the early stage of bone formation. H&E staining showed that autologous bone group was best. PRP and PRF group was better than control group. PRF showed similar effect comparing with PRP. From this, we can conclude that PRF can substitute for PRP.

가토 두개골 결손부에 이식된 ${\beta}-TCP$의 골치유 과정에서 PRP의 효과에 관한 연구

이성훈,황경균,박창주,임병섭,조정연,백승삼,심광섭,Lee, Soung-Hoon,Hwang, Kyung-Gyun,Park, Chang-Joo,Lim, Byung-Sup,Cho, Jung-Yeon,Paik, Seung-Sam,Shim, Kwang-Sup 대한악안면성형재건외과학회 2006 Maxillofacial Plastic Reconstructive Surgery Vol.28 No.5

Purpose : Platelet rich plasma (PRP) is an autologous material with many growth factors, such as BMPs, PDGF, $TGF-{\beta}_1$, $TGF-{\beta}_2$, VEGF, and IGF, facilitating bone healing process. The prominent osteoconductive activity and the biodegradable nature of beta-tricalciumphosphate (${\beta}-TCP$) for bone grafts in animal experiments have been reported. The purpose of this study was to evaluate the effect of PRP on the osteogenesis of ${\beta}-TCP$. Materials & Methods : Two artificial calvarial bone defects were made in 32 rabbits which were divided into 2 groups. In one group of 16 rabbits, autogenous bone / ${\beta}-TCP$ was grafted on each side of cranial bone defect. In the other group of 16 rabbits, mixture of ${\beta}-TCP$ and PRP / PRP alone was grafted on each side of the cranial bone defect. The animals were sacrificed at 2, 4, 8, and 12 weeks after surgery. The specimens were harvested and examined histologically and immunohistochemically by the expression of BMP2/4/7, PDGF, VEGF and $TGF-{\beta}_1$. Results : The mean volume of new bone formation was significantly higher at 4, 8, 12 weeks in autogenous graft than that in ${\beta}-TCP$. The BMP2/4 expression was significantly higher at 4 weeks in autogenous bone graft and at 4 weeks in mixture of ${\beta}-TCP$ and PRP and at 12 weeks in ${\beta}-TCP$. The expression of BMP7, PDGF, VEGF and $TGF-{\beta}_1$ showed no significant difference in autogenous, ${\beta}-TCP$, mixture of ${\beta}-TCP$ and PRP, and PRP alone during grafted bone regeneration. Conclusion : The results showed that PRP had no additional value in promoting healing process of ${\beta}-TCP$ grafts.

Curcumin Induces Apoptosis in Human Colorectal Carcinoma (HCT-15) Cells by Regulating Expression of Prp4 and p53

Adeeb Shehzad,이영섭,이재태,허태린 한국분자세포생물학회 2013 Molecules and cells Vol.35 No.6

Curcumin (diferuloylmethane), the yellow pigment of turmeric, is one of the most commonly used and exten-sively studied phytochemicals due to its pleiotropic effects in several human cancers. In the current study, the therapeutic efficacy of curcumin was investigated in human colorectal carcinoma HCT-15 cells. Curcumin inhibited HCT-15 cells proliferation and induced apoptosis in a dose- and time-dependent manner. Hoechst 33342 and DCFHDA staining revealed morpho-logical and biochemical features of apoptosis as well as ROS generation in HCT-15 cells treated with 30 and 50 M curcumin. Over-expression of pre-mRNA processing factor 4B (Prp4B) and p53 mutations have been reported as hallmarks of cancer cells. Western blot analysis revealed that curcumin treatment activated caspase-3 and decreased expression of p53 and Prp4B in a time-dependent manner. Transfection of HCT-15 cells with Prp4B clone perturbed the growth inhibition induced by 30 M curcumin. Fractionation of cells revealed increased accumulation of Prp4B in the nucleus, following its translocation from the cytoplasm. To further evaluate the underlying mechanism and survival effect of Prp4B, we generated siRNA-Prp4B HCT15 clones. Knockdown of Prp4B with siRNA diminished the protective effects of Prp4B against curcumin-induced apoptosis. These results suggest a possible underlying molecular mecha-nism in which Prp4B over-expression and activity are closely associated with the survival and regulation of apoptotic events in human colon cancer HCT-15 cells.

Decursin negatively regulates LPS-induced upregulation of the TLR4 and JNK signaling stimulated by the expression of PRP4 in vitro

Muhammad Bilal Ahmed,Salman Ul Islam,이영섭 한국통합생물학회 2020 Animal cells and systems Vol.24 No.1

The current investigation was carried out to analyze the correlation of bacterial lipopolysaccharide (LPS) and pre-mRNA processing factor 4B (PRP4) in inducing inflammatory response and cell actin cytoskeleton rearrangement in macrophages (Raw 264.7) and colorectal (HCT116) as well as skin cancer (B16-F10) cells. Cell lines were stimulated with LPS, and the expression of PRP4 as well as pro-inflammatory cytokines and proteins like IL-6, IL-1β, TLR4, and NF-κB were assayed. The results demonstrated that LPS markedly increased the expression of PRP4, IL-6, IL-1β, TLR4, and NF-κB in the cells. LPS and PRP4 concomitantly altered the morphology of cells from an aggregated, flattened shape to a round shape. Decursin, a pyranocoumarin from Angelica gigas, inhibited the LPS and PRP4-induced inflammatory response, and reversed the induction of morphological changes. Finally, we established a possible link of LPS with TLR4 and JNK signaling, through which it activated PRP4. Our study provides molecular insights for LPS and PRP4-related pathogenesis and a basis for developing new strategies against metastasis in colorectal cancer and skin melanoma. Our study emphasizes that decursin may be an effective treatment strategy for various cancers in which LPS and PRP4 perform a critical role in inducing inflammatory response and morphological changes leading to cell survival and protection against anti-cancer drugs.

Generation of ovine recombinant prion protein (25-232): Characterisation via anti-PrP monoclonal antibodies and CD spectroscopy

Yang, Su-Jeong,Thackray, Alana,Bujdoso, Raymond The Korean Society of Veterinary Service 2005 韓國家畜衛生學會誌 Vol.28 No.4

In prion pathogenesis, the structural conversion of the cellular prion protein $(PrP^c)$ to its abnormal isomer $(PrP^{Sc})$ is believed to be a major event. The susceptibility or resistance to natural sheep scrapie is associated with polymorphisms of host PrP gene (PRNP) at amino acid residues 136, to a lesser extent 154. The 112 residue in ovine PrP displays a natural polymorphism, Methionine to Threonine, which has not been thoroughly investigated. However the cell-free conversion assay showed that ARQ with Thr112 $(T_{112}ARQ)^{1)}$ presents lower convertibility to $PrP^{Sc}$than wild type ARQ $(M_{112}ARQ)$ [1] In this study we generated ovine recombinant PrPs of 112 allelic variants by metal chelate affinity chromatography and cation exchange chromatography. The final purity of the ovine PrP ARQ was more than $95\%$. These variants showed similar immunoreactivity against anti-PrP monoclonal antibodies in Western blot and ELISA. The refolded $M_{112}ARQ$ and $M_{112}ARQ$ presented the secondary structural content to similar extent via CD spectroscopy analysis. The inherited structural features of $M_{112}ARQ$ and $M_{112}ARQ$ under the different biophysical conditions are in the middle of investigation.