Knockdown Of Polo-like Kinase 1 Research Articles

Role of PLK1/NUMB/NOTCH in epithelial-mesenchymal transition in human melanoma

Polo-like kinase 1 (PLK1), a serine/threonine kinase, is overexpressed in melanoma and its expression has been associated with poor disease prognosis. PLK1 has been shown to interact with NUMB, a NOTCH antagonist. However, the exact role of PLK1, NUMB, and NOTCH signaling in epithelial-mesenchymal transition (EMT) in melanoma progression is unclear. In this study, Affymetrix microarray analysis was performed to determine differentially expressed genes following shRNA-mediated knockdown of PLK1 in human melanoma cells that showed significant modulations in EMT and metastasis-related genes. Using multiple PLK1-modulated melanoma cell lines, we found that PLK1 is involved in the regulation of cell migration, invasion, and EMT via its kinase activity and NOTCH activation. In vitro kinase assay and mass spectrometry analysis demonstrated a previously unknown PLK1 phosphorylation site (Ser413) on NUMB. Overexpression of non-phosphorylatable (S413A) and phosphomimetic (S413D) mutants of NUMB in melanoma cells implicated the involvement of NUMB-S413 phosphorylation in cell migration and invasion, which was independent of NOTCH activation. To determine the clinical relevance of these findings, immunohistochemistry was performed using melanoma tissue microarray, which indicated a strong positive correlation between PLK1 and N-cadherin, a protein required for successful EMT. These findings were supported by TCGA analysis, where expression of high PLK1 with low NUMB or high NOTCH or N-cadherin showed a significant decrease in survival of melanoma patients. Overall, these results suggest a potential role of PLK1 in EMT, migration, and invasion of melanoma cells. Our findings support the therapeutic targeting of PLK1, NUMB, and NOTCH for melanoma management.

MicroRNA-23a-3p promotes macrophage M1 polarization and aggravates lipopolysaccharide-induced acute lung injury by regulating PLK1/STAT1/STAT3 signalling.

Macrophage polarization is an important effector process in acute lung injury (ALI) induced by sepsis. MicroRNAs (miRNAs) have emerged as important players in regulating ALI process. Here, we showed that elevated microRNA-23a-3p (miR-23a-3p) promoted LPS-induced macrophage polarization and ALI in mice, while inhibition of miR-23a-3p led to reduced macrophage response and ameliorated ALI inflammation. Mechanically, miR-23a-3p regulated macrophage M1 polarization through targeting polo-like kinase 1 (PLK1). PLK1 was downregulated in LPS-treated macrophages and ALI mouse lung tissues. Knockdown of PLK1 increased macrophage M1 polarization through promoting STAT1/STAT3 activation, while overexpression of PLK1 reduced macrophage immune response. Collectively, our results reveal a key miRNA regulon that regulates macrophage polarization for LPS-induced immune response.

Activation of AMPK suppresses S1P-induced airway smooth muscle cells proliferation and its potential mechanisms

The aims of the present study were to investigate the signaling mechanisms for sphingosine-1-phosphate (S1P)-induced airway smooth muscle cells (ASMCs) proliferation and to explore the effect of activation of adenosine monophosphate-activated protein kinase (AMPK) on S1P-induced ASMCs proliferation and its underlying mechanisms. S1P phosphorylated signal transducer and activator of transcription 3 (STAT3) through binding to S1PR2/3, and this further sequentially up-regulated polo-like kinase 1 (PLK1) and inhibitor of differentiation 2 (ID2) protein expression. Pretreatment of cells with S1PR2 antagonist JTE-013, S1PR3 antagonist CAY-10444, knockdown of STAT3, PLK1 and ID2 attenuated S1P-triggered ASMCs proliferation. In addition, activation of AMPK by metformin inhibited S1P-induced ASMCs proliferation by suppressing STAT3 phosphorylation and therefore suppression of PLK1 and ID2 protein expression. Our study suggests that S1P promotes ASMCs proliferation by stimulating S1PR2/3/STAT3/PLK1/ID2 axis, and activation of AMPK suppresses ASMCs proliferation by targeting on STAT3 signaling pathway. Activation of AMPK might benefit asthma by inhibiting airway remodeling.

Inhibition of Polo-like kinase 1 (PLK1) facilitates the elimination of HIV-1 viral reservoirs in CD4+ T cells ex vivo.

Although combination antiretroviral therapy is effective in controlling HIV-1 infection, latent HIV-1 proviruses cannot be eliminated. HIV-1 reactivation induced by the mere use of latency-reversing agents is insufficient to render death of reservoir cells, indicating that certain intrinsic survival mechanisms exist. We report that Polo-like kinase 1 (PLK1) plays a critical role in survival of CD4+ T cells that undergo HIV-1 reactivation from latency or de novo infection. PLK1 is elevated in both scenarios, which requires HIV-1 Nef. HIV-1 enhances PLK1 SUMOylation, causing its nuclear translocation and protein stabilization. Inhibition or knockdown of PLK1 markedly facilitates death of HIV-1-infected CD4+ T cells. Furthermore, PLK1 inhibitors strikingly reduce the size of HIV-1 latent reservoirs in primary CD4+ T cells. Our findings demonstrate that HIV-1 infection hijacks PLK1 to prevent cell death induced by viral cytopathic effects, and that PLK1 is a promising target for chemical "killing" of HIV-1 reservoir cells.

Therapeutic potential of PLK1 inhibition in triple-negative breast cancer

Triple negative breast cancer (TNBC) is responsible for significant number of breast cancer-associated deaths because of lacking of successful molecular-targeted therapy. To explore a therapeutic target for TNBC, we performed a siRNA-mediated knockdown screening and identified Polo-like kinase 1 (PLK1) as a potential therapeutic target for TNBC. Knockdown of PLK1 as well as a small compound inhibitor for PLK1, BI-2536, induced G2/M arrest and created polyploid cell population, shown by increased DNA content and nuclear size. Inhibition of PLK1 eventually triggered apoptosis in multiple TNBC cell lines. In addition, we confirmed that PLK1 was significantly overexpressed in the tissues from TNBC patients compared with the tissues of normal mammary glands and benign breast tumors. Our data indicated that PLK1 plays a pivotal role in the regulation of mitosis of TNBC cells. Although future in vivo studies are warranted, targeting PLK1 by a selective inhibitor such as BI-2536 can be an attractive molecular-targeted therapy for TNBC.

Antileukemia Effects of Notch-Mediated Inhibition of Oncogenic PLK1 in B-Cell Acute Lymphoblastic Leukemia.

In B-cell acute lymphoblastic leukemia (B-ALL), activation of Notch signaling leads to cell-cycle arrest and apoptosis. We aimed to harness knowledge acquired by understanding a mechanism of Notch-induced cell death to elucidate a therapeutically viable target in B-ALL. To this end, we identified that Notch activation suppresses Polo-like kinase 1 (PLK1) in a B-ALL-specific manner. We identified that PLK1 is expressed in all subsets of B-ALL and is highest in Philadelphia-like (Ph-like) ALL, a high-risk subtype of disease. We biochemically delineated a mechanism of Notch-induced PLK1 downregulation that elucidated stark regulation of p53 in this setting. Our findings identified a novel posttranslational cascade initiated by Notch in which CHFR was activated via PARP1-mediated PARylation, resulting in ubiquitination and degradation of PLK1. This led to hypophosphorylation of MDM2Ser260, culminating in p53 stabilization and upregulation of BAX. shRNA knockdown or pharmacologic inhibition of PLK1 using BI2536 or BI6727 (volasertib) in B-ALL cell lines and patient samples led to p53 stabilization and cell death. These effects were seen in primary human B-ALL samples in vitro and in patient-derived xenograft models in vivo These results highlight PLK1 as a viable therapeutic target in B-ALL. Efficacy of clinically relevant PLK1 inhibitors in B-ALL patient-derived xenograft mouse models suggests that use of these agents may be tailored as an additional therapeutic strategy in future clinical studies.

Participation of PLK1 and FOXM1 in the hyperplastic proliferation of pulmonary artery smooth muscle cells in pulmonary arterial hypertension.

Vascular smooth muscle cells from the pulmonary arteries (HPASMC) of subjects with pulmonary arterial hypertension (PAH) exhibit hyperplastic growth. The PAH HPASMC display an increased sensitivity to fetal bovine serum (FBS) and undergo growth at a very low, 0.2%, FBS concentration. On the other hand, normal HPASMC (obtained from non-PAH donors) do not proliferate at low FBS (0.2%). A previous genomic study suggested that the nuclear factor, FOXM1 and the polo like kinase 1 (PLK1) are involved in promoting this hyperplastic growth of the PAH HPASMC. Here we find that limiting the action of FOXM1 or PLK1 not only restricts the hyperplastic proliferation of the PAH HPASMC but also modulates the FBS stimulated growth of normal HPASMC. The PAH HPASMC exhibit significantly elevated PLK1 and FOXM1 expression and decreased p27 (quiescence protein) levels compared to normal HPASMC. Regulation of the expression of FOXM1 and PLK1 is accompanied by the regulation of downstream expression of cell cycle components, Aurora B, cyclin B1 and cyclin D1. Expression of these cell cycle components is reversed by the knockdown of FOXM1 or PLK1 expression/activity. Furthermore, the knockdown of PLK1 expression lowers the protein level of FOXM1. On the other hand, inhibiting the action of FOXO1, a growth inhibitor, further increases the expression of FOXM1 in PAH HPASMC. Although PLK1 and FOXM1 clearly participate in PAH HPASMC hyperplasia, at this time it is not clear whether their increased activity is the primary driver of the hyperplastic behavior of the PAH HPASMC or merely a component of the pathway(s) leading to this response.

Kinome-Wide Screening with Small Interfering RNA Identified Polo-like Kinase 1 as a Key Regulator of Proliferation in Oral Cancer Cells.

Oral squamous cell carcinoma (OSCC) is one of the major leading causes of cancer-related death worldwide, with limited effective markers for diagnosis and therapy, which has caused a low overall survival rate in the past decades. Kinases play important roles in tumor development and malignancy in various types of cancer. However, little is known about the role of kinases in OSCC cells. In this study, an arrayed kinome small interfering RNA (siRNA) library was used to screen oral cancer cell lines and counter assayed with normal fibroblast cells to identify the genes required for cancer cell proliferation. We found that polo-like kinase 1 (PLK1) was one of the most potent genes required for OSCC cell proliferation. The knockdown of PLK1 with a siRNA or antisense oligonucleotide (ASO) consistently diminished cyclin-B1 (CCNB1) expression/phosphorylation and the G2-M phase transition. Similar effects were observed in cells treated with the PLK1 kinase inhibitor BI6727. Besides, The Cancer Genome Atlas (TCGA) analysis revealed that PLK1 was elevated in tumor tissues and associated with short survival in patients with OSCC. We also found that PLK1 expression was highly correlated with the expression of its downstream effector, CCNB1, in patients with OSCC. Coexpression of the two genes resulted in a poor prognosis of OSCC patients, particularly those in the advanced stages of OSCC. Taken together, our results suggest that PLK1 might be a diagnostic or therapeutic marker for OSCC.

Correction: Kinome-wide Functional Screen Identifies Role of PLK1 in Hormone-Independent, ER-Positive Breast Cancer.

Estrogen receptor α (ER)-positive breast cancers initially respond to antiestrogens but eventually become estrogen-independent and recur. ER+ breast cancer cells resistant to long-term estrogen deprivation (LTED) exhibit hormone-independent ER transcriptional activity and growth. A kinome-wide siRNA screen using a library targeting 720 kinases identified Polo-like kinase 1 (PLK1) as one of the top genes whose downregulation resulted in inhibition of estrogen-independent ER transcriptional activity and growth of LTED cells. High PLK1 mRNA and protein correlated with a high Ki67 score in primary ER+ breast cancers after treatment with the aromatase inhibitor letrozole. RNAi-mediated knockdown of PLK1 inhibited ER expression, estrogen-independent growth and ER transcription in MCF7 and HCC1428 LTED cells. Pharmacological inhibition of PLK1 with volasertib, a small molecule ATP-competitive PLK1 inhibitor, decreased LTED cell growth, ER transcriptional activity and ER expression. Volasertib in combination with the ER antagonist, fulvestrant, decreased MCF7 xenograft growth in ovariectomized mice more potently than each drug alone. JUNB, a component of the AP-1 complex, was expressed 16-fold higher in MCF7/LTED compared to parental MCF7 cells. Further, JUNB and BCL2L1 (which encodes anti-apoptotic BCL-xL) mRNA levels were markedly reduced upon volasertib treatment in MCF7/LTED cells while they were increased in parental MCF7 cells. Finally, JUNB knockdown decreased ER expression and transcriptional activity in MCF7/LTED cells, suggesting that PLK1 drives ER expression and estrogen-independent growth via JUNB. These data support a critical role of PLK1 in acquired hormone-independent growth of ER+ human breast cancer and is therefore a promising target in tumors that have escaped estrogen deprivation therapy.

Knockdown of PLK1 inhibits invasion and promotes apoptosis in glioma cells through regulating autophagy.

Polo like kinase 1 (PLK1), an oncogene, is a ubiquitously expressed serine/threonine protein kinase. We aimed at investigating the role of PLK1 in glioma. Clinical glioma specimens were obtained from Zhejiang Hospital (Hangzhou, Zhejiang, China). The mRNA and protein levels of PLK1 in glioma tissues and different glioma cells were analyzed by Real-time PCR and Western blot, respectively. The expression of PLK1 protein in glioma tissues was also determined by immunohistochemistry staining. Then, the effect of PLK1 on cell proliferation and apoptosis of U251 and U87 cells was analyzed by using CCK-8 assay and Annexin V/PI staining, respectively. Furthermore, the migration and invasion of glioma cells were examined by transwell assay. Finally, the protein levels of autophagy indicators LC3-II, ATG5 and p-p70 S6 in U251 and U87 cells were detected by Western blot, and the expression of E-cadherin, vimentin and MMP9 and apoptosis associated indicators Bax, cleaved caspase-3 and Bcl-2 in U251 cells were also determined using Western blot. PLK1 was upregulated in glioma tissues and cells. Knockdown of PLK1 significantly inhibited cell proliferation, migration, invasion, and induced apoptosis of U87 and U251 glioma cells. Furthermore, the data demonstrated that knockdown of PLK1 significantly elevated expression of cleaved caspase-3, BIM, BAX, and E-cadherin, and reduced expression of MMP9, ATG5 and LC3-II in U251 and U87 cells. Additionally, we found that knockdown of PLK1 can inhibit autophagy in glioma cells. Knockdown of PLK1 can inhibit the glioma development by suppressing the autophagy and enhancing the apoptosis of glioma cells. PLK1 may be a potential therapeutic target in gliomas.

Abstract 3816: Mutations of the lim protein ajuba mediate sensitivity of head and neck squamous cell carcinoma to treatment with cell cycle inhibitors

Abstract The genomic alterations identified in head and neck squamous cell carcinoma (HNSCC) tumors have not resulted in any changes in clinical care, making the development of biomarker-driven targeted therapy for HNSCC a major translational gap in knowledge. To fill this gap, we used 59 molecularly characterized HNSCC cell lines and found that mutations of AJUBA,SMAD4 and RAS predicted sensitivity and resistance to treatment with inhibitors of polo-like kinase 1 (PLK1), checkpoint kinases 1 and 2, and WEE1. Inhibition or knockdown of PLK1 led to cell-cycle arrest at the G2/M transition and apoptosis in sensitive cell lines and decreased tumor growth in an orthotopic AJUBA-mutant HNSCC mouse model. AJUBA protein expression was undetectable in most AJUBA-mutant HNSCC cell lines, and total PLK1 protein expression was increased in cell lines wild-type for AJUBA. Exogenous expression of wild-type AJUBA in an AJUBA-mutant cell line partially rescued the phenotype of PLK1 inhibitor-induced apoptosis and decreased PLK1 substrate inhibition, suggesting a threshold effect in which higher drug doses are required to affect PLK1 substrate inhibition. PLK1 inhibition was an effective therapy for HNSCC in vitro and in vivo. However, biomarkers to guide such therapy are lacking. We identified AJUBA, SMAD4 and RAS mutations as potential candidate biomarkers of response of HNSCC to treatment with these mitotic inhibitors. Citation Format: Ming Zhang, Singh Ratnakar, Shaohua Peng, Mazumdar Tuhina, Shen Li, Pan Tong, Curtis Pickering, Jeffrey N. Myers, Jing Wang, Faye M. Johnson. Mutations of the lim protein ajuba mediate sensitivity of head and neck squamous cell carcinoma to treatment with cell cycle inhibitors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3816. doi:10.1158/1538-7445.AM2017-3816

Mutations of the LIM protein AJUBA mediate sensitivity of head and neck squamous cell carcinoma to treatment with cell-cycle inhibitors

The genomic alterations identified in head and neck squamous cell carcinoma (HNSCC) tumors have not resulted in any changes in clinical care, making the development of biomarker-driven targeted therapy for HNSCC a major translational gap in knowledge. To fill this gap, we used 59 molecularly characterized HNSCC cell lines and found that mutations of AJUBA, SMAD4 and RAS predicted sensitivity and resistance to treatment with inhibitors of polo-like kinase 1 (PLK1), checkpoint kinases 1 and 2, and WEE1. Inhibition or knockdown of PLK1 led to cell-cycle arrest at the G2/M transition and apoptosis in sensitive cell lines and decreased tumor growth in an orthotopic AJUBA-mutant HNSCC mouse model. AJUBA protein expression was undetectable in most AJUBA-mutant HNSCC cell lines, and total PLK1 and Bora protein expression were decreased. Exogenous expression of wild-type AJUBA in an AJUBA-mutant cell line partially rescued the phenotype of PLK1 inhibitor-induced apoptosis and decreased PLK1 substrate inhibition, suggesting a threshold effect in which higher drug doses are required to affect PLK1 substrate inhibition. PLK1 inhibition was an effective therapy for HNSCC in vitro and in vivo. However, biomarkers to guide such therapy are lacking. We identified AJUBA, SMAD4 and RAS mutations as potential candidate biomarkers of response of HNSCC to treatment with these mitotic inhibitors.

FOXM1 participates in PLK1-regulated cell cycle progression in renal cell cancer cells.

The regulation of entry into and progression through mitosis is important for cell proliferation. Polo-like kinase 1 (PLK1) is involved in multiple stages of mitosis. Forkhead box protein M1 (FOXM1) has multiple functions in tumorigenesis and, in elevated levels, is frequently associated with cancer progression. The present study reports that FOXM1, a substrate of PLK1, controls the transcription mechanism that mediates the PLK1-dependent regulation of the cell cycle. The present study investigated the expression of PLK1 and FOXM1 in the clear renal cell carcinoma 769-P and ACHN cell lines, and indicated that the expression of PLK1 and FOXM1 are correlated in human renal cell cancer cell lines and that the suppression of PLK1 may decrease the expression of FOXM1. The knockdown of FOXM1 or PLK1 in renal cell cancer cell lines caused cell cycle progression to be blocked. As a result, the present study indicated the involvement of FOXM1 in PLK1-regulated cell cycle progression.

JNK-associated Leucine Zipper Protein Functions as a Docking Platform for Polo-like Kinase 1 and Regulation of the Associating Transcription Factor Forkhead Box Protein K1

JLP (JNK-associated leucine zipper protein) is a scaffolding protein that interacts with various signaling proteins associated with coordinated regulation of cellular process such as endocytosis, motility, neurite outgrowth, cell proliferation, and apoptosis. Here we identified PLK1 (Polo-like kinase 1) as a novel interaction partner of JLP through mass spectrometric approaches. Our results indicate that JLP is phospho-primed by PLK1 on Thr-351, which is recognized by the Polo box domain of PLK1 leading to phosphorylation of JLP at additional sites. Stable isotope labeling by amino acids in cell culture and quantitative LC-MS/MS analysis was performed to identify PLK1-dependent JLP-interacting proteins. Treatment of cells with the PLK1 kinase inhibitor BI2536 suppressed binding of the Forkhead box protein K1 (FOXK1) transcriptional repressor to JLP. JLP was found to interact with PLK1 and FOXK1 during mitosis. Moreover, knockdown of PLK1 affected the interaction between JLP and FOXK1. FOXK1 is a known transcriptional repressor of the CDK inhibitor p21/WAF1, and knockdown of JLP resulted in increased FOXK1 protein levels and a reduction of p21 transcript levels. Our results suggest a novel mechanism by which FOXK1 protein levels and activity are regulated by associating with JLP and PLK1.

MiR-296-5p suppresses cell viability by directly targeting PLK1 in non-small cell lung cancer.

Polo-like kinase 1 (PLK1), a critical kinase for mitotic progression, is overexpressed in a wide range of cancers. MicroRNAs (miRNAs) are a class of small non-coding RNA molecules and proposed to play important roles in the regulation of tumor progression and invasion. However, the relationship between PLK1 and miRNAs have remained unclear. In the present study, the association between PLK1 and miR-296-5p was investigated. The upregulation of PLK1 mRNA expression levels combined with the downregulation of miR-296-5p levels were detected in both non-small cell lung cancer (NSCLC) tissues and cell lines. Functional studies showed that knockdown of PLK1 by siRNA inhibited NSCLC cells proliferation. Impressively, overexpression of miR-296-5p showed the same phenocopy as the effect of PLK1 knockdown in NSCLC cells, indicating that PLK1 was a major target of miR-296-5p. Furthermore, using western blot analysis and luciferase reporter assay, PLK1 protein expression was proved to be regulated by miR-296-5p through binding to the putative binding sites in its 3'-untranslated region (3'-UTR). Taken together, the present study indicated that miR-296-5p regulated PLK1 expression and could function as a tumor suppressor in NSCLC progression, which provides a potential target for gene therapy of NSCLC.

Kinome-wide functional screen identifies role of PLK1 in hormone-independent, ER-positive breast cancer.

Estrogen receptor (ER) α-positive breast cancers initially respond to antiestrogens but eventually become estrogen independent and recur. ER(+) breast cancer cells resistant to long-term estrogen deprivation (LTED) exhibit hormone-independent ER transcriptional activity and growth. A kinome-wide siRNA screen using a library targeting 720 kinases identified Polo-like kinase 1 (PLK1) as one of the top genes whose downregulation resulted in inhibition of estrogen-independent ER transcriptional activity and growth of LTED cells. High PLK1 mRNA and protein correlated with a high Ki-67 score in primary ER(+) breast cancers after treatment with the aromatase inhibitor letrozole. RNAi-mediated knockdown of PLK1 inhibited ER expression, estrogen-independent growth, and ER transcription in MCF7 and HCC1428 LTED cells. Pharmacologic inhibition of PLK1 with volasertib, a small-molecule ATP-competitive PLK1 inhibitor, decreased LTED cell growth, ER transcriptional activity, and ER expression. Volasertib in combination with the ER antagonist, fulvestrant, decreased MCF7 xenograft growth in ovariectomized mice more potently than each drug alone. JUNB, a component of the AP-1 complex, was expressed 16-fold higher in MCF7/LTED compared with parental MCF7 cells. Furthermore, JUNB and BCL2L1 (which encodes antiapoptotic BCL-xL) mRNA levels were markedly reduced upon volasertib treatment in MCF7/LTED cells, while they were increased in parental MCF7 cells. Finally, JUNB knockdown decreased ER expression and transcriptional activity in MCF7/LTED cells, suggesting that PLK1 drives ER expression and estrogen-independent growth via JUNB. These data support a critical role of PLK1 in acquired hormone-independent growth of ER(+) human breast cancer and is therefore a promising target in tumors that have escaped estrogen deprivation therapy.

A promising gene delivery system developed from PEGylated MoS2 nanosheets for gene therapy.

A new class of two-dimensional (2D) nanomaterial, transition metal dichalcogenides (TMDCs) such as MoS2, MoSe2, WS2, and WSe2 which have fantastic physical and chemical properties, has drawn tremendous attention in different fields recently. Herein, we for the first time take advantage of the great potential of MoS2 with well-engineered surface as a novel type of 2D nanocarriers for gene delivery and therapy of cancer. In our system, positively charged MoS2-PEG-PEI is synthesized with lipoic acid-modified polyethylene glycol (LA-PEG) and branched polyethylenimine (PEI). The amino end of positively charged nanomaterials can bind to the negatively charged small interfering RNA (siRNA). After detection of physical and chemical characteristics of the nanomaterial, cell toxicity was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Polo-like kinase 1 (PLK1) was investigated as a well-known oncogene, which was a critical regulator of cell cycle transmission at multiple levels. Through knockdown of PLK1 with siRNA carried by novel nanovector, qPCR and Western blot were used to measure the interfering efficiency; apoptosis assay was used to detect the transfection effect of PLK1. All results showed that the novel nanocarrier revealed good biocompatibility, reduced cytotoxicity, as well as high gene-carrying ability without serum interference, thus would have great potential for gene delivery and therapy.

PSA-responsive and PSMA-mediated multifunctional liposomes for targeted therapy of prostate cancer

In the hormone-refractory stage of prostate cancer (PC), the expression of prostate-specific antigen (PSA) and prostate-specific membrane antigen (PSMA) often remains highly active. Accumulating studies have demonstrated that these two proteins are attractive targets for specific delivery of functional molecules to advanced PC, not merely as potential sensitive markers for PC detection. In this study, we constructed a dual-modified liposome that incorporated PSA-responsive and PSMA-mediated liposomes and potentially offers double selectivity for PC. The folate moiety binds quickly to PSMA-positive tumors, and the PSA-responsive moiety is cleaved by PSA that was enriched in tumor tissues. The activated liposomes (folate and cell-penetrating peptides dual-modifications) are subsequently taken up by the tumor cells via polyarginine's penetrating effects and receptor-mediated endocytosis. To corroborate these assumptions, a series of experiments were conducted, including PSA-responsive peptide hydrolysis kinetics, cellular uptake, internalization mechanism and escape from endosomes in PC-3 and/or 22Rv1 cells, biodistribution and antitumor activity of siRNA-loaded liposomes after systemic administration, gene silencing and cell apoptosis in vitro and in vivo. The results reveal that multivalent interactions play a key role in enhancing PC cell recognition and uptake while reducing nonspecific uptake. The dual-modified liposomes carrying small interfering RNA (siRNA) have significant advantages over the control liposomes, including single-modified (folate, CPP, PSA-responsive only) and non-modified liposomes. The dual-modified liposomes elevated cellular uptake, downregulated expression of polo-like kinase 1 (PLK-1) and augmented cell apoptosis in prostate tumor cells. The entry of the dual-modified liposomes into 22Rv1 cells occurred via multiple endocytic pathways, including clathrin-mediated endocytosis and macropinocytosis, followed by an effective endosomal escape of the entrapped siRNA into the cytoplasm. In vivo studies conducted on a 22Rv1 xenograft murine model demonstrated that the dual-modified liposomes demonstrated the maximized accumulation, retention and knockdown of PLK-1 in tumor cells, as well as the strongest inhibition of tumor growth and induction of tumor cell apoptosis. In terms of targeting capacity and therapeutic potency, the combination of a PSA-responsive and PSMA-mediated liposome presents a promising platform for therapy and diagnosis of PSMA/PSA-positive PC.

Enhanced chemosensitivity to CPT-11 in colorectal carcinoma xenografts by small hairpin RNA interference targeting PLK1

Commonly used drugs for the treatment of colon{} cancer patients like CPT-11 shows severe side effects or induces resistance in clinical settings. Thus, we analyzed a combination of PLK1 (polo-like kinase 1)-specific short hair RNA (shRNA), a potent tool to destroy mitosis in cancer cells, together with CPT-11 to enhance drug sensitivity. Cellular proliferation and apoptosis were determined in SW620 colorectal carcinoma cells. Knockdown of cellular PLK1 led to the decreased mRNA and PLK1 protein in RT-PCR and western blot assay. The viability declined (p<0.001) in MTT assay and colony formation assay, and the number of apoptotic cells was clearly increased (p<0.01) in flow cytometric analysis and Hoechst 33258 staining compared with control cells after incubation with PLK1-specific shRNA and SN-38. We found the level of cleaved PARP was also increased in vitro. In vivo, employment of shRNA targeting PLK1 improved the sensitivity to treat SW620 nude mouse model toward CPT-11. The combination therapy inhibited cellular proliferation and promoted apoptosis observed at the percentage of PCNA and caspase3 by immunohistochemistry, accompanied with TUNEL assay. As we expect, the combination treatment delayed tumor growth (p<0.01) and simultaneously reduced tumor weight (p<0.01) compared with control group. Taken together, combination of PLK1-specific shRNA interference with low-dose CPT-11 triggered a antitumor efficacy and represented a potential strategy to treat colon cancer.

Efficient Inhibition of Human Colorectal Carcinoma Growth by RNA Interference Targeting Polo-Like Kinase 1 In Vitro and In Vivo

Polo-like kinase 1 (PLK1) showing a high expression in various kinds of tumors is considered a candidate target for cancer therapy. The aim of our study was to explore the effects of silencing PLK1 gene on human colorectal carcinoma cell line HCT-116 in vitro and in vivo. In vitro, the plasmids generating short hairpin RNA (shRNA)-targeting PLK1 were transfected into HCT-116 by using FugeneHD reagent, and the silencing potency was measured by RT-PCR, western blot, flow cytometry, and Caspase-Glo 3/7 assay, respectively. In vivo, the growth inhibition capacity of PLK1-shRNA on HCT-116 xenograft was measured in nude mice. Then, the silencing effect of PLK1 was analyzed by RT-PCR, western blot, and immunohistochemistry, respectively. Apoptosis, angiogenesis, and proliferation in tumor tissues were measured by TUNEL, CD31, and PCNA stain, respectively. The RNA interference targeting PLK1 significantly decreased the expression of PLK1 in vitro. More importantly, anti-PLK1 treatment in HCT-116 xenograft decreased tumor weight by 81.58% compared with the control group (p<0.001), accompanied with decreased PLK1 mRNA and protein expression, increased cell apoptosis, and reduced angiogenesis and proliferation (p<0.001). Our study showed that knockdown of PLK1 by shRNA might be the potential therapeutic approach against human colorectal carcinoma.