CD15 expression has been used for years to confirm the diagnosis of Hodgkin's disease (HD... more CD15 expression has been used for years to confirm the diagnosis of Hodgkin's disease (HD). Little is, however, known on the relevance of the CD15 antigen to the pathobiology of the disease and there is conflicting evidence as to the prognostic value of its expression. To investigate the significance of the differential expression of CD15 in Hodgkin's disease, a retrospective study of 102 patients with "classical" Hodgkin's disease was performed. Immunohistochemical studies were carried out using antibodies against two types of CD15: non-sialylated CD15 (LeuM1 and 80H5) and sialylated CD15 (FH6 and CSLEX1). Cases that were negative for non-sialylated CD15 or positive for the sialylated variant were stained again following neuraminidase pretreatment. The cohort included 27 patients in whom sequential biopsies were available. Both CD15 expression in its non-sialylated form and absence of sialyl-CD15 expression correlate with a favorable outcome. Subsequent biopsies show a preferential expression of sialyl-CD15, notably in bone marrow metastases. Our findings suggest that, in the progression of HD towards a widely disseminated disease, the LewisX moiety of the CD15 antigen on the tumor cells acquires a sialyl-group. This change may confer on the tumor cells the capacity to metastasize.
Cardiovascular morbidity is the leading cause of death of obstructive sleep apnea (OSA) syndrome ... more Cardiovascular morbidity is the leading cause of death of obstructive sleep apnea (OSA) syndrome patients. Nocturnal airway obstruction is associated with intermittent hypoxia (IH). In our previous work with cell lines, incubation with sera from OSA patients induced changes in cell morphology, NF-κB activation and decreased viability. A decrease in beating rate, contraction amplitude and a reduction in intracellular calcium signaling was also observed in human cardiomyocytes differentiated from human embryonic stem cells (hESC-CMs). We expanded these observations using a new controlled IH in vitro system on beating hESC-CMs. The Oxy-Cycler system was programed to generate IH cycles. Following IH, we detected the activation of Hif-1α as an indicator of hypoxia and nuclear NF-κB p65 and p50 subunits, representing pro-inflammatory activity. We also detected the secretion of inflammatory cytokines, such as MIF, PAI-1, MCP-1 and CXCL1, and demonstrated a decrease in beating rate of hESC-...
Marine natural products have recently been recognized as a promising source of NF-κB inhibitors. ... more Marine natural products have recently been recognized as a promising source of NF-κB inhibitors. In this study, extracts of bacterial isolates from the coral Favis sp. were screened for NF-κB modulatory capability using an NF-κB luciferase reporter gene assay. The bacterial extracts had variable effects on NF-κB activity: from the 39 extracts tested, only one exhibited significant NF-κB inhibition and two caused NF-κB up regulation. In addition, we showed that coral bacteria New-33 extract inhibits NF-κB alternative pathway subunits in a non-cytotoxic manner. HPLC analysis confirmed the presence of a low molecular mass compound and NCBI megaBLAST revealed that the New-33 16S rRNA gene sequences are similar to Vibrio mediterranei. This specific modulation of the NF-κB by alternative pathway could contribute to therapeutic implications and also raises questions about host-symbiont interaction in the marine environment.
Introduction There are cardiovascular morbid effects of OSA in adults and children. We have previ... more Introduction There are cardiovascular morbid effects of OSA in adults and children. We have previously reported over-expression of Nuclear-Factor-κappa B in tonsils of children with OSA, and NF-κB activation after exposure to sera of children with OSA. In order to investigate NF-kB activation and the physiology of cardiomyocytes of human origin (CM’s), we have established an ex-vivo model where CM’s derived from human embryonic stem cells H-9.1 clone (hES) and from Induced pluripotent stem cells (iPSC) are either incubated with human sera, or exposed to intermittent hypoxia(IH) or room air (RA). Methods Serum samples were drawn following overnight polysomnography (PSG) from children with OSA (AHI>5) or control (AHI<1). Differentiated human cardiomyocytes were incubated with 5% OSA sera or control. Average cell beating/min was determined and NF-kB p50 and p65 cytoplasmic or nuclear localization were detected by immunofluorescence (operetta). CM’s were exposed to either RA or to...
The expression of a cell death-inducing gene, Bax, was investigated in 52 cases of Hodgkin's ... more The expression of a cell death-inducing gene, Bax, was investigated in 52 cases of Hodgkin's disease in parallel with Epstein-Barr virus and was compared with the immunodetection of other apoptosis-regulating proteins, Mcl-1, Bcl-2, and Bcl-x. Bax immunostaining was found in 92% of the cases, among them 28% with a strong signal in more than 75% of the Reed-Sternberg cells. Mcl-1 was positive in 80% of the cases, whereas Bcl-2 and Bcl-x were found in 53% and 88% of the cases, respectively. Of 48 (89%) Bax-positive tumors, 43 were found to express apoptosis-inhibiting proteins such as Mcl-1 or Bcl-2. With the exception of 1 case, all Bax-positive tumors also expressed either Bcl- 2, Bcl-x, Mcl-1, or combinations of these anti-apoptotic proteins. No correlation was found between Bax expression and the presence of apoptotic cells as detected by morphology and the in situ 3′ OH-DNA end- labeling technique. Our findings show that the apoptosis-inducing gene Bax expression is frequentl...
The Hodgkin-Reed-Sternberg (HRS) malignant cells in Hodgkin's lymphoma (HL) originate from ge... more The Hodgkin-Reed-Sternberg (HRS) malignant cells in Hodgkin's lymphoma (HL) originate from germinal center B lymphocytes that did not undergo apoptosis. Protein Kinase C (PKC), a family of serine/threonine kinases, plays a crucial role in signal transduction modulating cell growth, differentiation and apoptosis. Here, we report the expression of PKC isoforms in two HL-derived cell lines, L428 and KMH2 and their correlation with drug resistance to CPT and doxorubicin. Among the PKC isoforms examined, only PKCeta and PKCbetaII were preferentially expressed in the drug resistant L428 cells. We have shown correlation between the response to apoptosis of L428 and KMH2 cells and PKCeta expression in these cell lines. In order to directly demonstrate a role for PKCeta in apoptosis, its expression was knocked-down by siRNA in the resistant L428 cells. Downregulation of PKCeta rendered L428 cells more sensitive to doxorubicin and CPT. Furthermore, PKCeta knocked-down cells showed increas...
In recent years several reports have been published describing dogs' ability to detect, by sc... more In recent years several reports have been published describing dogs' ability to detect, by scent, patients with cancer. This ability is based on the sniffing of volatile organic elements that are secreted by malignant cells or react to them. To evaluate the ability of trained dogs to detect breast cancer cell cultures (MCF7) compared to the control pseudo-normal keratinocyte cell line (HaCaT), and to detect melanoma (BG) and type 2 epithelial lung carcinoma (A549) malignant cell cultures to which they were not previously exposed in the course of their training. Cell cultures were prepared in a standard manner. Two Belgian Shepherd dogs were trained and then tested in a single-blind test (for dogs and trainers) on their ability to detect the "target specimen," a MCF7 breast cancer cell culture. Following this, the ability of the dogs to detect cancer cell cultures that they were not previously exposed to (i.e., A549, BG) was tested. In each test round, four specimens pl...
BACKGROUND Malignant melanoma resistance to chemotherapy remains a major limitation to treatment.... more BACKGROUND Malignant melanoma resistance to chemotherapy remains a major limitation to treatment. Our aim was to identify genes associated with drug resistance, in order to better understand the molecular events underlying the drug-resistant phenotype. MATERIALS AND METHODS A human melanoma cell line and its drug-resistant variants obtained by selection with MNNG or 6-thioguanine were used. Alterations in gene expression were characterized by differential display reverse transcription-polymerase chain reaction (DDRT-PCR). Prominent mRNA fragments present in selected variants and not in the parental cells were identified and characterized by cloning and sequencing. Differential expression was confirmed by real-time RT-PCR. RESULTS Three functionally distinct transcriptional products were demonstrated: the chaperonin subunit TCP 1-zeta-6A (CCT6A), the hyaluronan receptor CD44 and LPPR-2, the lipid phosphate phosphatase-related protein type-2. CONCLUSION Genes with altered expression w...
Different parts of Nuphar lutea L. (yellow water lily) have been used to treat several inflammato... more Different parts of Nuphar lutea L. (yellow water lily) have been used to treat several inflammatory and pathogen-related diseases. It has shown that Nuphar lutea extracts (NUP) are active against various pathogens including bacteria, fungi, and leishmanial parasites. In an effort to detect novel therapeutic agents against negative-stranded RNA (- RNA) viruses, we have tested the effect of a partially-purified alkaloid mixture of Nuphar lutea leaves on the measles virus (MV). The MV vaccine’s Edmonston strain was used to acutely or persistently infect cells. The levels of several MV proteins were detected by a Western blot and immunocytochemistry. Viral RNAs were quantitated by qRT-PCR. Virus infectivity was monitored by infecting African green monkey kidney VERO cells’ monolayers. We showed that NUP protected cells from acute infection. Decreases in the MV P-, N-, and V-proteins were observed in persistently infected cells and the amount of infective virus released was reduced as co...
Hodgkin9s lymphoma (HL), a B cell originated malignancy of the immune system, is one of the most ... more Hodgkin9s lymphoma (HL), a B cell originated malignancy of the immune system, is one of the most frequent lymphomas in the Western world. Although the rate of cure is high, about 20-35% of patients relapse, and about half of them eventually die of the disease or treatment-related late toxicities and secondary malignancies. Conceptually novel treatment strategies are thus needed, particularly for this category of patients. The malignant cells, called Hodgkin and Reed-Sternberg (HRS) cells, only make up 1-2% of the total tumor cellularity; the remaining mass comprising a mixed infiltrate population is thought to be recruited to the lymph node by HRS-driven pro-inflammatory signals. The origin of the pro-inflammatory signals remains elusive, and represents a critical gap in our knowledge of the pathogenesis of HL. We propose that a sub-population of HRS cells, which we call herein large RS cells, have characteristics of senescent cells, and, thus produce large amounts of inflammatory mediators (the so-called “senescence-associated secretory phenotype”, or SASP). Senescence is characterized by permanent cell cycle arrest and loss of proliferative capacity, despite continued viability and metabolic activity. We show that the senescence markers the cell cycle inhibitors p16INK4a and p21Cip1 are expressed in large RS cells in all HL biopsies examined. Moreover, the large RS cells are negative for Ki-67 staining, demonstrating that these cells have ceased to proliferate. We also show that large, ‘RS-like’ cells in a HL-derived line, L428, stained for the characteristic senescence marker β-galactosidase (β-gal). Oxidative stress and chemotherapy increased the proportion of β-gal positive large RS cells. Furthermore, we show that the large RS cells secrete high levels of cytokines. We suggest that these senescent RS cells may be responsible, at least in part, for creating a pro-inflammatory microenvironment, promoting HL pathogenesis, and mediating chemo-resistance in relapsed disease. Understanding the pathways important for the establishment of senescence in HRS cells, as well gaining insight into targetable mechanisms for the eradication of these cells, will provide new therapeutic approaches for HL patients with recurrent or chemo-resistant disease. Citation Format: Jacob Gopas, Udi Zurgil, Estee Zeev, Daniel Benharoch, Jodi Ezratty, Giora Shubinsky, Etta Livneh. Cellular senescence in Hodgkin9s lymphoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1263. doi:10.1158/1538-7445.AM2015-1263
The Hodgkin-Reed-Sternberg (HRS) malignant cells in Hodgkin's lymphoma (HL) originate from germin... more The Hodgkin-Reed-Sternberg (HRS) malignant cells in Hodgkin's lymphoma (HL) originate from germinal center B lymphocytes that did not undergo apoptosis. Protein Kinase C (PKC), a family of serine/threonine kinases, plays a crucial role in signal transduction modulating cell growth, differentiation and apoptosis. Here, we report the expression of PKC isoforms in two HL-derived cell lines, L428 and KMH2 and their correlation with drug resistance to CPT and doxorubicin. Among the PKC isoforms examined, only PKCη and PKCbII were preferentially expressed in the drug resistant L428 cells. We have shown correlation between the response to apoptosis of L428 and KMH2 cells and PKCη expression in these cell lines. In order to directly demonstrate a role for PKCη in apoptosis, its expression was knocked-down by siRNA in the resistant L428 cells. Downregulation of PKCη rendered L428 cells more sensitive to doxorubicin and CPT. Furthermore, PKCη knocked-down cells showed increased PARP-1 cleavage, cytochrome c release and caspase 7 activation. It appears that PKCη functions as an anti-apoptotic protein in HL-derived cell lines, and as we show here that it is also expressed in HRS of HL biopsies, it may have therapeutic relevance in HL. Thus, PKCη could provide a new target aimed to reduce resistance to anti-cancer treatments of HL and other cancer patients.
Biochemical and biophysical research communications, Jan 26, 2011
The nuclear factor κB (NF-κB) family of transcription factors participates in the regulation of g... more The nuclear factor κB (NF-κB) family of transcription factors participates in the regulation of genes involved in innate- and adaptive-immune responses, cell death and inflammation. The involvement of the Protein kinase C (PKC) family in the regulation of NF-κB in inflammation and immune-related signaling has been extensively studied. However, not much is known on the role of PKC in NF-κB regulation in response to DNA damage. Here we demonstrate for the first time that PKC-eta (PKCη) regulates NF-κB upstream signaling by activating the IκB kinase (IKK) and the degradation of IκB. Furthermore, PKCη enhances the nuclear translocation and transactivation of NF-κB under non-stressed conditions and in response to the anticancer drug camptothecin. We and others have previously shown that PKCη confers protection against DNA damage-induced apoptosis. Our present study suggests that PKCη is involved in NF-κB signaling leading to drug resistance.
licenses/by-nc-nd/3.0/). Reproduction is permitted for personal, noncommercial use, provided that... more licenses/by-nc-nd/3.0/). Reproduction is permitted for personal, noncommercial use, provided that the article is in whole, unmodified, and properly cited. Received: 2012.06.10; Accepted: 2012.07.21; Published: 2012.07.24 We recognized a few possible complications of classical Hodgkin lymphoma therapy in a cohort of 209 patients: 8 developed a primary refractory disease (primary progression), 36 showed an early relapse and 21 showed a late relapse. Sialyl-CD15 expression in Hodgkin-Reed-Sternberg cells was significantly more positive in primary refractory Hodgkin lymphoma, which confirms our previously published findings. Bcl-2 showed a significantly lower level of expression in primary refractory disease than in the other follow-up groups. This is in contrast with a previous finding of Bcl-2, associated with a poor prognosis in primary refractory illness. Another category of variables, old age and advanced stages, was significantly different in the various complications but this fin...
Water lily (Nuphar) bioactive extracts have been widely used in traditional medicine owing to the... more Water lily (Nuphar) bioactive extracts have been widely used in traditional medicine owing to their multiple applications against human ailments. Phyto-active Nuphar extracts and their purified and synthetic derivatives have attracted the attention of ethnobotanists and biochemists. Here, we report that 6,6′-dihydroxythiobinupharidine (DTBN), purified from extracts of Nuphar lutea (L.) Sm. leaves, is an effective inhibitor of the kinase activity of members of the protein kinase C (PKC) family using in vitro and in silico approaches. We demonstrate that members of the conventional subfamily of PKCs, PKCα and PKCγ, were more sensitive to DTBN inhibition as compared to novel or atypical PKCs. Molecular docking analysis demonstrated the interaction of DTBN, with the kinase domain of PKCs depicting the best affinity towards conventional PKCs, in accordance with our in vitro kinase activity data. The current study reveals novel targets for DTBN activity, functioning as an inhibitor for PK...
Measles virus (MV) infects a variety of lymphoid and non-lymphoid peripheral organs. However, in ... more Measles virus (MV) infects a variety of lymphoid and non-lymphoid peripheral organs. However, in rare cases, the virus can persistently infect cells within the central nervous system. Although some of the factors that allow MV to persist are known, the contribution of host cell-encoded microRNAs (miRNA) have not been described. MiRNAs are a class of noncoding RNAs transcribed from genomes of all multicellular organisms and some viruses, which regulate gene expression in a sequence-specific manner. We have studied the contribution of host cell-encoded miRNAs to the establishment of MV persistent infection in human neuroblastoma cells. Persistent MV infection was accompanied by differences in the expression profile and levels of several host cell-encoded microRNAs as compared to uninfected cells. MV persistence infection of a human neuroblastoma cell line (UKF-NB-MV), exhibit high miRNA-124 expression, and reduced expression of cyclin dependent kinase 6 (CDK6), a known target of miRNA-124, resulting in slower cell division but not cell death. By contrast, acute MV infection of UKF-NB cells did not result in increased miRNA-124 levels or CDK6 reduction. Ectopic overexpression of miRNA-124 affected cell viability only in UKF-NB-MV cells, causing cell death; implying that miRNA-124 over expression can sensitize cells to death only in the presence of MV persistent infection. To determine if miRNA-124 directly contributes to the establishment of MV persistence, UKF-NB cells overexpressing miRNA-124 were acutely infected, resulting in establishment of persistently infected colonies. We propose that miRNA-124 triggers a CDK6-dependent decrease in cell proliferation, which facilitates the establishment of MV persistence in neuroblastoma cells. To our knowledge, this is the first report to describe the role of a specific miRNA in MV persistence.
Nuphar lutea L. SM., leaf and rhizome extracts (NUP), contain nupharidines as active components. ... more Nuphar lutea L. SM., leaf and rhizome extracts (NUP), contain nupharidines as active components. Nupharidines belong to the sesquiterpene lactones class of a naturally occurring plant terpenoids. This family of compounds has gained considerable interest for treating infection, inflammation and cancer. NF-κB is a central, downstream regulator of inflammation, cell proliferation and apoptosis. In our previous work we demonstrated strong inhibition of NF-κB activity and induction of apoptosis by NUP. In addition, NUP exhibited anti-inflammatory properties and partial protection from LPS-induced septic shock by modulating ERK pathway and cytokine secretion in macrophages. In the present study, we examined the effect of NUP in a B16 melanoma experimental murine lung metastasis model and its ability to affect the ERK and NF-κB pathways in variety of cell lines. We showed that NUP and cisplatin combined treatment was synergistic and reduced the lung metastatic load. In addition NUP treatment inhibited TNFα-induced IκBα degradation and NF-κB nuclear translocation. We also observed that NUP induced ERK activation. Furthermore, ERK inhibition prevented NF-κB inactivation by NUP. Overall, our work implies that co-administration of NF-κB inhibitors such as NUP, with standard anti-cancer drugs, may act as "sensitizers" for more effective chemotherapy.
CD15 expression has been used for years to confirm the diagnosis of Hodgkin&#39;s disease (HD... more CD15 expression has been used for years to confirm the diagnosis of Hodgkin&#39;s disease (HD). Little is, however, known on the relevance of the CD15 antigen to the pathobiology of the disease and there is conflicting evidence as to the prognostic value of its expression. To investigate the significance of the differential expression of CD15 in Hodgkin&#39;s disease, a retrospective study of 102 patients with &quot;classical&quot; Hodgkin&#39;s disease was performed. Immunohistochemical studies were carried out using antibodies against two types of CD15: non-sialylated CD15 (LeuM1 and 80H5) and sialylated CD15 (FH6 and CSLEX1). Cases that were negative for non-sialylated CD15 or positive for the sialylated variant were stained again following neuraminidase pretreatment. The cohort included 27 patients in whom sequential biopsies were available. Both CD15 expression in its non-sialylated form and absence of sialyl-CD15 expression correlate with a favorable outcome. Subsequent biopsies show a preferential expression of sialyl-CD15, notably in bone marrow metastases. Our findings suggest that, in the progression of HD towards a widely disseminated disease, the LewisX moiety of the CD15 antigen on the tumor cells acquires a sialyl-group. This change may confer on the tumor cells the capacity to metastasize.
Cardiovascular morbidity is the leading cause of death of obstructive sleep apnea (OSA) syndrome ... more Cardiovascular morbidity is the leading cause of death of obstructive sleep apnea (OSA) syndrome patients. Nocturnal airway obstruction is associated with intermittent hypoxia (IH). In our previous work with cell lines, incubation with sera from OSA patients induced changes in cell morphology, NF-κB activation and decreased viability. A decrease in beating rate, contraction amplitude and a reduction in intracellular calcium signaling was also observed in human cardiomyocytes differentiated from human embryonic stem cells (hESC-CMs). We expanded these observations using a new controlled IH in vitro system on beating hESC-CMs. The Oxy-Cycler system was programed to generate IH cycles. Following IH, we detected the activation of Hif-1α as an indicator of hypoxia and nuclear NF-κB p65 and p50 subunits, representing pro-inflammatory activity. We also detected the secretion of inflammatory cytokines, such as MIF, PAI-1, MCP-1 and CXCL1, and demonstrated a decrease in beating rate of hESC-...
Marine natural products have recently been recognized as a promising source of NF-κB inhibitors. ... more Marine natural products have recently been recognized as a promising source of NF-κB inhibitors. In this study, extracts of bacterial isolates from the coral Favis sp. were screened for NF-κB modulatory capability using an NF-κB luciferase reporter gene assay. The bacterial extracts had variable effects on NF-κB activity: from the 39 extracts tested, only one exhibited significant NF-κB inhibition and two caused NF-κB up regulation. In addition, we showed that coral bacteria New-33 extract inhibits NF-κB alternative pathway subunits in a non-cytotoxic manner. HPLC analysis confirmed the presence of a low molecular mass compound and NCBI megaBLAST revealed that the New-33 16S rRNA gene sequences are similar to Vibrio mediterranei. This specific modulation of the NF-κB by alternative pathway could contribute to therapeutic implications and also raises questions about host-symbiont interaction in the marine environment.
Introduction There are cardiovascular morbid effects of OSA in adults and children. We have previ... more Introduction There are cardiovascular morbid effects of OSA in adults and children. We have previously reported over-expression of Nuclear-Factor-κappa B in tonsils of children with OSA, and NF-κB activation after exposure to sera of children with OSA. In order to investigate NF-kB activation and the physiology of cardiomyocytes of human origin (CM’s), we have established an ex-vivo model where CM’s derived from human embryonic stem cells H-9.1 clone (hES) and from Induced pluripotent stem cells (iPSC) are either incubated with human sera, or exposed to intermittent hypoxia(IH) or room air (RA). Methods Serum samples were drawn following overnight polysomnography (PSG) from children with OSA (AHI>5) or control (AHI<1). Differentiated human cardiomyocytes were incubated with 5% OSA sera or control. Average cell beating/min was determined and NF-kB p50 and p65 cytoplasmic or nuclear localization were detected by immunofluorescence (operetta). CM’s were exposed to either RA or to...
The expression of a cell death-inducing gene, Bax, was investigated in 52 cases of Hodgkin's ... more The expression of a cell death-inducing gene, Bax, was investigated in 52 cases of Hodgkin's disease in parallel with Epstein-Barr virus and was compared with the immunodetection of other apoptosis-regulating proteins, Mcl-1, Bcl-2, and Bcl-x. Bax immunostaining was found in 92% of the cases, among them 28% with a strong signal in more than 75% of the Reed-Sternberg cells. Mcl-1 was positive in 80% of the cases, whereas Bcl-2 and Bcl-x were found in 53% and 88% of the cases, respectively. Of 48 (89%) Bax-positive tumors, 43 were found to express apoptosis-inhibiting proteins such as Mcl-1 or Bcl-2. With the exception of 1 case, all Bax-positive tumors also expressed either Bcl- 2, Bcl-x, Mcl-1, or combinations of these anti-apoptotic proteins. No correlation was found between Bax expression and the presence of apoptotic cells as detected by morphology and the in situ 3′ OH-DNA end- labeling technique. Our findings show that the apoptosis-inducing gene Bax expression is frequentl...
The Hodgkin-Reed-Sternberg (HRS) malignant cells in Hodgkin's lymphoma (HL) originate from ge... more The Hodgkin-Reed-Sternberg (HRS) malignant cells in Hodgkin's lymphoma (HL) originate from germinal center B lymphocytes that did not undergo apoptosis. Protein Kinase C (PKC), a family of serine/threonine kinases, plays a crucial role in signal transduction modulating cell growth, differentiation and apoptosis. Here, we report the expression of PKC isoforms in two HL-derived cell lines, L428 and KMH2 and their correlation with drug resistance to CPT and doxorubicin. Among the PKC isoforms examined, only PKCeta and PKCbetaII were preferentially expressed in the drug resistant L428 cells. We have shown correlation between the response to apoptosis of L428 and KMH2 cells and PKCeta expression in these cell lines. In order to directly demonstrate a role for PKCeta in apoptosis, its expression was knocked-down by siRNA in the resistant L428 cells. Downregulation of PKCeta rendered L428 cells more sensitive to doxorubicin and CPT. Furthermore, PKCeta knocked-down cells showed increas...
In recent years several reports have been published describing dogs' ability to detect, by sc... more In recent years several reports have been published describing dogs' ability to detect, by scent, patients with cancer. This ability is based on the sniffing of volatile organic elements that are secreted by malignant cells or react to them. To evaluate the ability of trained dogs to detect breast cancer cell cultures (MCF7) compared to the control pseudo-normal keratinocyte cell line (HaCaT), and to detect melanoma (BG) and type 2 epithelial lung carcinoma (A549) malignant cell cultures to which they were not previously exposed in the course of their training. Cell cultures were prepared in a standard manner. Two Belgian Shepherd dogs were trained and then tested in a single-blind test (for dogs and trainers) on their ability to detect the "target specimen," a MCF7 breast cancer cell culture. Following this, the ability of the dogs to detect cancer cell cultures that they were not previously exposed to (i.e., A549, BG) was tested. In each test round, four specimens pl...
BACKGROUND Malignant melanoma resistance to chemotherapy remains a major limitation to treatment.... more BACKGROUND Malignant melanoma resistance to chemotherapy remains a major limitation to treatment. Our aim was to identify genes associated with drug resistance, in order to better understand the molecular events underlying the drug-resistant phenotype. MATERIALS AND METHODS A human melanoma cell line and its drug-resistant variants obtained by selection with MNNG or 6-thioguanine were used. Alterations in gene expression were characterized by differential display reverse transcription-polymerase chain reaction (DDRT-PCR). Prominent mRNA fragments present in selected variants and not in the parental cells were identified and characterized by cloning and sequencing. Differential expression was confirmed by real-time RT-PCR. RESULTS Three functionally distinct transcriptional products were demonstrated: the chaperonin subunit TCP 1-zeta-6A (CCT6A), the hyaluronan receptor CD44 and LPPR-2, the lipid phosphate phosphatase-related protein type-2. CONCLUSION Genes with altered expression w...
Different parts of Nuphar lutea L. (yellow water lily) have been used to treat several inflammato... more Different parts of Nuphar lutea L. (yellow water lily) have been used to treat several inflammatory and pathogen-related diseases. It has shown that Nuphar lutea extracts (NUP) are active against various pathogens including bacteria, fungi, and leishmanial parasites. In an effort to detect novel therapeutic agents against negative-stranded RNA (- RNA) viruses, we have tested the effect of a partially-purified alkaloid mixture of Nuphar lutea leaves on the measles virus (MV). The MV vaccine’s Edmonston strain was used to acutely or persistently infect cells. The levels of several MV proteins were detected by a Western blot and immunocytochemistry. Viral RNAs were quantitated by qRT-PCR. Virus infectivity was monitored by infecting African green monkey kidney VERO cells’ monolayers. We showed that NUP protected cells from acute infection. Decreases in the MV P-, N-, and V-proteins were observed in persistently infected cells and the amount of infective virus released was reduced as co...
Hodgkin9s lymphoma (HL), a B cell originated malignancy of the immune system, is one of the most ... more Hodgkin9s lymphoma (HL), a B cell originated malignancy of the immune system, is one of the most frequent lymphomas in the Western world. Although the rate of cure is high, about 20-35% of patients relapse, and about half of them eventually die of the disease or treatment-related late toxicities and secondary malignancies. Conceptually novel treatment strategies are thus needed, particularly for this category of patients. The malignant cells, called Hodgkin and Reed-Sternberg (HRS) cells, only make up 1-2% of the total tumor cellularity; the remaining mass comprising a mixed infiltrate population is thought to be recruited to the lymph node by HRS-driven pro-inflammatory signals. The origin of the pro-inflammatory signals remains elusive, and represents a critical gap in our knowledge of the pathogenesis of HL. We propose that a sub-population of HRS cells, which we call herein large RS cells, have characteristics of senescent cells, and, thus produce large amounts of inflammatory mediators (the so-called “senescence-associated secretory phenotype”, or SASP). Senescence is characterized by permanent cell cycle arrest and loss of proliferative capacity, despite continued viability and metabolic activity. We show that the senescence markers the cell cycle inhibitors p16INK4a and p21Cip1 are expressed in large RS cells in all HL biopsies examined. Moreover, the large RS cells are negative for Ki-67 staining, demonstrating that these cells have ceased to proliferate. We also show that large, ‘RS-like’ cells in a HL-derived line, L428, stained for the characteristic senescence marker β-galactosidase (β-gal). Oxidative stress and chemotherapy increased the proportion of β-gal positive large RS cells. Furthermore, we show that the large RS cells secrete high levels of cytokines. We suggest that these senescent RS cells may be responsible, at least in part, for creating a pro-inflammatory microenvironment, promoting HL pathogenesis, and mediating chemo-resistance in relapsed disease. Understanding the pathways important for the establishment of senescence in HRS cells, as well gaining insight into targetable mechanisms for the eradication of these cells, will provide new therapeutic approaches for HL patients with recurrent or chemo-resistant disease. Citation Format: Jacob Gopas, Udi Zurgil, Estee Zeev, Daniel Benharoch, Jodi Ezratty, Giora Shubinsky, Etta Livneh. Cellular senescence in Hodgkin9s lymphoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1263. doi:10.1158/1538-7445.AM2015-1263
The Hodgkin-Reed-Sternberg (HRS) malignant cells in Hodgkin's lymphoma (HL) originate from germin... more The Hodgkin-Reed-Sternberg (HRS) malignant cells in Hodgkin's lymphoma (HL) originate from germinal center B lymphocytes that did not undergo apoptosis. Protein Kinase C (PKC), a family of serine/threonine kinases, plays a crucial role in signal transduction modulating cell growth, differentiation and apoptosis. Here, we report the expression of PKC isoforms in two HL-derived cell lines, L428 and KMH2 and their correlation with drug resistance to CPT and doxorubicin. Among the PKC isoforms examined, only PKCη and PKCbII were preferentially expressed in the drug resistant L428 cells. We have shown correlation between the response to apoptosis of L428 and KMH2 cells and PKCη expression in these cell lines. In order to directly demonstrate a role for PKCη in apoptosis, its expression was knocked-down by siRNA in the resistant L428 cells. Downregulation of PKCη rendered L428 cells more sensitive to doxorubicin and CPT. Furthermore, PKCη knocked-down cells showed increased PARP-1 cleavage, cytochrome c release and caspase 7 activation. It appears that PKCη functions as an anti-apoptotic protein in HL-derived cell lines, and as we show here that it is also expressed in HRS of HL biopsies, it may have therapeutic relevance in HL. Thus, PKCη could provide a new target aimed to reduce resistance to anti-cancer treatments of HL and other cancer patients.
Biochemical and biophysical research communications, Jan 26, 2011
The nuclear factor κB (NF-κB) family of transcription factors participates in the regulation of g... more The nuclear factor κB (NF-κB) family of transcription factors participates in the regulation of genes involved in innate- and adaptive-immune responses, cell death and inflammation. The involvement of the Protein kinase C (PKC) family in the regulation of NF-κB in inflammation and immune-related signaling has been extensively studied. However, not much is known on the role of PKC in NF-κB regulation in response to DNA damage. Here we demonstrate for the first time that PKC-eta (PKCη) regulates NF-κB upstream signaling by activating the IκB kinase (IKK) and the degradation of IκB. Furthermore, PKCη enhances the nuclear translocation and transactivation of NF-κB under non-stressed conditions and in response to the anticancer drug camptothecin. We and others have previously shown that PKCη confers protection against DNA damage-induced apoptosis. Our present study suggests that PKCη is involved in NF-κB signaling leading to drug resistance.
licenses/by-nc-nd/3.0/). Reproduction is permitted for personal, noncommercial use, provided that... more licenses/by-nc-nd/3.0/). Reproduction is permitted for personal, noncommercial use, provided that the article is in whole, unmodified, and properly cited. Received: 2012.06.10; Accepted: 2012.07.21; Published: 2012.07.24 We recognized a few possible complications of classical Hodgkin lymphoma therapy in a cohort of 209 patients: 8 developed a primary refractory disease (primary progression), 36 showed an early relapse and 21 showed a late relapse. Sialyl-CD15 expression in Hodgkin-Reed-Sternberg cells was significantly more positive in primary refractory Hodgkin lymphoma, which confirms our previously published findings. Bcl-2 showed a significantly lower level of expression in primary refractory disease than in the other follow-up groups. This is in contrast with a previous finding of Bcl-2, associated with a poor prognosis in primary refractory illness. Another category of variables, old age and advanced stages, was significantly different in the various complications but this fin...
Water lily (Nuphar) bioactive extracts have been widely used in traditional medicine owing to the... more Water lily (Nuphar) bioactive extracts have been widely used in traditional medicine owing to their multiple applications against human ailments. Phyto-active Nuphar extracts and their purified and synthetic derivatives have attracted the attention of ethnobotanists and biochemists. Here, we report that 6,6′-dihydroxythiobinupharidine (DTBN), purified from extracts of Nuphar lutea (L.) Sm. leaves, is an effective inhibitor of the kinase activity of members of the protein kinase C (PKC) family using in vitro and in silico approaches. We demonstrate that members of the conventional subfamily of PKCs, PKCα and PKCγ, were more sensitive to DTBN inhibition as compared to novel or atypical PKCs. Molecular docking analysis demonstrated the interaction of DTBN, with the kinase domain of PKCs depicting the best affinity towards conventional PKCs, in accordance with our in vitro kinase activity data. The current study reveals novel targets for DTBN activity, functioning as an inhibitor for PK...
Measles virus (MV) infects a variety of lymphoid and non-lymphoid peripheral organs. However, in ... more Measles virus (MV) infects a variety of lymphoid and non-lymphoid peripheral organs. However, in rare cases, the virus can persistently infect cells within the central nervous system. Although some of the factors that allow MV to persist are known, the contribution of host cell-encoded microRNAs (miRNA) have not been described. MiRNAs are a class of noncoding RNAs transcribed from genomes of all multicellular organisms and some viruses, which regulate gene expression in a sequence-specific manner. We have studied the contribution of host cell-encoded miRNAs to the establishment of MV persistent infection in human neuroblastoma cells. Persistent MV infection was accompanied by differences in the expression profile and levels of several host cell-encoded microRNAs as compared to uninfected cells. MV persistence infection of a human neuroblastoma cell line (UKF-NB-MV), exhibit high miRNA-124 expression, and reduced expression of cyclin dependent kinase 6 (CDK6), a known target of miRNA-124, resulting in slower cell division but not cell death. By contrast, acute MV infection of UKF-NB cells did not result in increased miRNA-124 levels or CDK6 reduction. Ectopic overexpression of miRNA-124 affected cell viability only in UKF-NB-MV cells, causing cell death; implying that miRNA-124 over expression can sensitize cells to death only in the presence of MV persistent infection. To determine if miRNA-124 directly contributes to the establishment of MV persistence, UKF-NB cells overexpressing miRNA-124 were acutely infected, resulting in establishment of persistently infected colonies. We propose that miRNA-124 triggers a CDK6-dependent decrease in cell proliferation, which facilitates the establishment of MV persistence in neuroblastoma cells. To our knowledge, this is the first report to describe the role of a specific miRNA in MV persistence.
Nuphar lutea L. SM., leaf and rhizome extracts (NUP), contain nupharidines as active components. ... more Nuphar lutea L. SM., leaf and rhizome extracts (NUP), contain nupharidines as active components. Nupharidines belong to the sesquiterpene lactones class of a naturally occurring plant terpenoids. This family of compounds has gained considerable interest for treating infection, inflammation and cancer. NF-κB is a central, downstream regulator of inflammation, cell proliferation and apoptosis. In our previous work we demonstrated strong inhibition of NF-κB activity and induction of apoptosis by NUP. In addition, NUP exhibited anti-inflammatory properties and partial protection from LPS-induced septic shock by modulating ERK pathway and cytokine secretion in macrophages. In the present study, we examined the effect of NUP in a B16 melanoma experimental murine lung metastasis model and its ability to affect the ERK and NF-κB pathways in variety of cell lines. We showed that NUP and cisplatin combined treatment was synergistic and reduced the lung metastatic load. In addition NUP treatment inhibited TNFα-induced IκBα degradation and NF-κB nuclear translocation. We also observed that NUP induced ERK activation. Furthermore, ERK inhibition prevented NF-κB inactivation by NUP. Overall, our work implies that co-administration of NF-κB inhibitors such as NUP, with standard anti-cancer drugs, may act as "sensitizers" for more effective chemotherapy.
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