Asymmetric silicon phthalocyanine based nanoparticle with spatiotemporally targeting of mitochondria for synergistic apoptosis-ferroptosis antitumor treatment.

A promising therapeutic strategy in cancer treatment merges photodynamic therapy (PDT) induced apoptosis with ferroptosis, a form of programmed cell death governed by iron-dependent lipid peroxidation. Given the pivotal role of mitochondria in ferroptosis, the development of photosensitizers that specifically provoke mitochondrial dysfunction and consequentially trigger ferroptosis via PDT is of significant interest. To this end, we have designed and synthesized a novel nanoparticle, termed FECTPN, tailored to address this requisite. FECTPN harnesses a trifecta of critical attributes: precision mitochondria targeting, photoactivation capability, pH-responsive drug release, and synergistic apoptosis-ferroptosis antitumor treatment. This nanoparticle was formulated by conjugating an asymmetric silicon phthalocyanine, Chol-SiPc-TPP, with the ferroptosis inducer Erastin onto a ferritin. The Chol-SiPc-TPP is a chemically crafted entity featuring cholesteryl (Chol) and triphenylphosphine (TPP) functionalities bonded axially to the silicon phthalocyanine, enhancing mitochondrial affinity and leading to effective PDT and subsequent apoptosis of cells. Upon cellular uptake, FECTPN preferentially localizes to mitochondria, facilitated by Chol-SiPc-TPP's targeting mechanics. Photoactivation induces the synchronized release of Chol-SiPc-TPP and Erastin in the mitochondria's alkaline domain, driving the escalation of both ROSs and lipid peroxidation. These processes culminate in elevated antitumor activity compared to the singular application of Chol-SiPc-TPP-mediated PDT. A notable observation is the pronounced enhancement in glutathione peroxidase-4 (GPX4) expression within MCF-7 cells treated with FECTPN and subjected to light exposure, reflecting intensified oxidative stress. This study offers compelling evidence that FECTPN can effectively induce ferroptosis and reinforces the paradigm of a synergistic apoptosis-ferroptosis pathway in cancer therapy, proposing a novel route for augmented antitumor treatments.

Death-associated protein kinase 1 suppresses hepatocellular carcinoma cell migration and invasion by upregulation of DEAD-box helicase 20.

Death-associated protein kinase 1 (DAPK) is a calcium/calmodulin kinase that plays a vital role as a suppressor gene in various cancers. Yet its role and target gene independent of p53 is still unknown in hepatocellular carcinoma (HCC). In this study, we discovered that DAPK suppressed HCC cell migration and invasion instead of proliferation or colony formation. Using a proteomics approach, we identified DEAD-box helicase 20 (DDX20) as an important downstream target of DAPK in HCC cells and critical for DAPK-mediated inhibition of HCC cell migration and invasion. Using integrin inhibitor RGD and GTPase activity assays, we discovered that DDX20 suppressed HCC cell migration and invasion through the CDC42-integrin pathway, which was previously reported as an important downstream pathway of DAPK in cancer. Further research using cycloheximide found that DAPK attenuates the proteasomal degradation of DDX20 protein, which is dependent on the kinase activity of DAPK. Our results shed light on new functions and regulation for both DAPK and DDX20 in carcinogenesis and identifies new potential therapeutic targets for HCC.

Distribution, Trafficking, and in Vitro Photodynamic Therapy Efficacy of Cholesterol Silicon(IV) Phthalocyanine and Its Nanoparticles in Breast Cancer Cells.

A cholesterol silicon(IV) phthalocyanine (Chol-Pc) and a water-soluble Chol-Pc based nanoparticle (DSPE@Chol-Pc), which was prepared using 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(poly(ethylene glycol))-2000] (DSPE-PEG2000) as a nanocarrier were developed. Chol-Pc readily distributed within the cholesterol-rich domains and was preferentially localized in the Golgi apparatus after being transported into the cells. The trafficking of DSPE@Chol-Pc in breast cancer cells was visualized by tracking the fluorescence of Chol-Pc and FITC-labeled DSPE-PEG2000 through two-photonic imaging in real-time. It was discovered that Chol-Pc disassociated from the DSPE-PEG2000 on the plasma membrane and traveled to the cholesterol-rich domains soon afterward. Both DSPE@Chol-Pc and Chol-Pc effectively mediated photodynamic therapy to kill the breast cancer cells. After light irradiation, we found that the organizations of clustered cholesterol-rich domains in cells were destroyed, presumably leading to the death of cells for photodynamic therapy. It should be noted that DSPE@Chol-Pc is highly soluble in aqueous solution and has strong red fluorescence under two-photon excitation. Thus, it could be an excellent probe for detecting cholesterol-rich domains and studying transport processes of cholesterol in living cells.

Evaluation of the prognostic and physiological functions of death associated protein kinase 1 in breast cancer.

Death associated protein kinase 1 (DAPK1) is a notable serine/threonine kinase involved in the regulation of multiple cellular pathways, including apoptosis and autophagy. Although DAPK1 is usually considered to be a tumor suppressor, it was previously reported to promote the viability of p53 mutant cancer cell lines and possess physiological oncogenic functions in breast cancer. However, the ability of endogenous DAPK1 to suppress breast cancer cell mobility has not been assessed. In the present study, the prognostic function of DAPK1 in a Chinese patient cohort was evaluated, and no significant association was observed between DAPK1 expression and patient survival or lymph node metastasis. In order to investigate the physiological function of endogenous DAPK1, stable inducible DAPK1 knockdown MCF7 and MDA-MB-231 cell lines were established. Consistent with previous studies, endogenous DAPK1 only regulated cell viability in p53 mutant MDA-MB-231 cells. However, knockdown of DAPK1 did not significantly affect cell motility of either MCF7 or MDA-MB-231 cells. Altogether, these results further explored the function of endogenous DAPK1 in breast cancer and may shed light in understanding the molecular signaling pathways regulating the physiological function of DAPK1.

The Roles of Protein Tyrosine Phosphatases in Hepatocellular Carcinoma.

The protein tyrosine phosphatase (PTP) family is involved in multiple cellular functions and plays an important role in various pathological and physiological processes. In many chronic diseases, for example cancer, PTP is a potential therapeutic target for cancer treatment. In the last two decades, dozens of PTP inhibitors which specifically target individual PTP molecules were developed as therapeutic agents. Hepatocellular carcinoma (HCC) is one of the most common malignant tumors and is the second most lethal cancer worldwide due to a lack of effective therapies. Recent studies have unveiled both oncogenic and tumor suppressive functions of PTP in HCC. Here, we review the current knowledge on the involvement of PTP in HCC and further discuss the possibility of targeting PTP in HCC.

Probing the biophysical properties of tumor cells during mitosis by atomic force microscopy.

Mitosis is an important physiological event accompanying with dramatic changes of cellar biophysical properties. Failure of mitosis results in cell death or chromosome aneuploidy. In this study, we used atomic force microscopy to probe and compare the biophysical properties of tumor cells at different stages during mitosis. The rounding forces of MCF-7 cells oscillated during mitosis. At anaphase, the average elasticity of cells was higher than that at other phases. Cholesterol depletion with M[Formula: see text]CD led to an increase in the average elasticity, whereas the average roughness of membrane surface decreased at the absence of cholesterol. Our study indicated that the distribution of actin filaments could affect the biophysical properties of tumor cells and cellular morphology during mitosis. Furthermore, the biophysical properties of tumor cells were also regulated by membrane cholesterol during mitosis. This work provides a new detection approach for monitoring tumor cell development at single cell level.

Ouabain targets the Na+/K+-ATPase α3 isoform to inhibit cancer cell proliferation and induce apoptosis.

Ouabain has been used for the treatment of heart failure and atrial fibrillation. Its potential anticancer effect has also attracted great interest. The aim of the present study was to evaluate the anticancer effect of ouabain and investigate its molecular target. The effects of ouabain on the viability of and induction of cellular death on OS-RC-2 renal cancer cells were examined using the MTT assay and acridine orange/ethidium bromide staining. The levels of Ca2+ and reactive oxygen species were determined using Fura-3-acetoxymethyl ester and dichloro-dihydro-fluorescein diacetate probes, respectively. Apoptosis was examined using annexin V-fluorescein isothiocyanate/propidium iodide staining and western blotting. The expression profile of the different Na+/K+-ATPase (NKA) isoforms in NCI-H446 small cell lung cancer cells was determined using immunocytochemistry and reverse transcription polymerase chain reaction analysis. In the present study, it was demonstrated that ouabain inhibited cancer cell proliferation and induced apoptosis while no significant difference in the expression of NKA α1 and α3 isoforms was detected following 48 h of ouabain treatment. Furthermore, expression of NKA α3 but not the α1 isoform was associated with ouabain sensitivity. The results of the present study indicated that ouabain targets the NKA α3 isoform, inhibits cancer cell proliferation and induces apoptosis.

Quantitative and correlation analysis of the DNA methylation and expression of DAPK in breast cancer.

BACKGROUND: Death-associated protein kinase 1 (DAPK) is an important tumor suppressor kinase involved in the regulation of multiple cellular activities such as apoptosis and autophagy. DNA methylation of DAPK gene was found in various types of cancers and often correlated with the clinicopathological characteristics. However, the mRNA and protein expression of DAPK in the same sample was rarely measured. Thus, it was unclear if the correlation between DAPK gene methylation and clinicopathological parameters was due to the loss of DAPK expression. METHODS: In this study, the DNA methylation rate, mRNA and protein expression of DAPK was quantitatively detected in 15 pairs of breast cancer patient samples including tumor (T) and adjacent non-tumor (N) tissues. RESULTS: The correlation between DNA methylation rate and mRNA expression, together with the correlation between mRNA and protein expression, was calculated. No correlation was observed between any levels using either the measurement value of each sample or the T/N ratio of each pair. DISCUSSION: These data suggested that the DNA methylation status of DAPK did not correlate well with its mRNA or protein expression. Extra caution is needed when interpreting the DNA methylation data of DAPK gene in clinical studies.

Digitoxin synergizes with sorafenib to inhibit hepatocelluar carcinoma cell growth without inhibiting cell migration.

Sorafenib is a chemotherapeutic agent approved for the treatment of hepatocellular carcinoma (HCC) in China. Digitoxin is a cardiotonic drug, which has been demonstrated to exhibit anticancer effects in a number of cancers, but not in HCC. The aim of the present study was to evaluate the combinational effect of sorafenib and digitoxin on the treatment of HCC and to investigate the relevant molecular mechanisms of action that underlie these effects. The proliferation, cell death and migration of HCC cell lines, HepG2 and BEL­7402, were examined using MTT, acridine orange/ethidium bromide staining and scratch wound healing assays, respectively. In addition, alterations in the expression of phosphorylated-extracellular signal-regulated kinase (ERK), hypoxia­inducible factor 1­α (HIF­1α), hypoxia­inducible factor 2­α (HIF­2α) and vascular endothelial growth factor (VEGF) were measured prior to and following drug application using western blot analysis. Digitoxin and sorafenib synergistically inhibited cell viability, but did not inhibit migration, which was potentially mediated by suppression of ERK and hypoxia signaling. In downstream signaling pathways, the activity of ERK was synergistically suppressed by combinatorial treatment of HepG2 and BEL­7402 cells with sorafenib and digitoxin. In addition, the expression of HIF­1α, HIF­2α and VEGF was synergistically downregulated by combinational treatment.

Triblock copolymers encapsulated poly (aryl benzyl ether) dendrimer zinc(II) phthalocyanine nanoparticles for enhancement in vitro photodynamic efficacy.

A novel series of nanoparticles formed via an electrostatic interaction between the periphery of negatively charged 1-2 generation aryl benzyl ether dendrimer zinc (II) phthalocyanines and positively charged poly(L-lysin) segment of triblock copolymer, poly(L-lysin)-block-poly(ethylene glycol)-block-poly(L-lysin), was developed for the use as an effective photosensitizers in photodynamic therapy. The dynamic light scattering, atomic force microscopy showed that two nanoparticles has a relevant size of 80-150nm. The photophysical properties and singlet oxygen quantum yields of free dendrimer phthalocyanines and nanoparticles exhibited generation dependence. The intracellular uptake of dendrimer phthalocyanines in Hela cells was significantly elevated as they were incorporated into the micelles, but was inversely correlated with the generation of dendrimer phthalocyanines. The photocytotoxicity of dendrimer phthalocyanines incorporated into polymeric micelles was also increased. The presence of nanoparticles induced efficient cell death. Using a mitochondrial-sepcific dye rhodamine 123 (Rh123), our fluorescence microscopic result indicated that nanoparticles localized to the mitochondria.

The prognostic significance of PD-L1 in bladder cancer.

Immunotherapy is a promising strategy for the treatment of various types of cancer. An antibody that targets programmed death ligand-1 (PD-L1) pathway has been shown to be active towards various types of cancer, including melanoma and lung cancer. MPDL3280A, an anti-PD-L1 antibody, has shown clear clinical activity in PD-L1-overexpressing bladder cancer with an objective response rate of 40-50%, resulting in a breakthrough therapy designation granted by FDA. These events pronounce the importance of targeting the PD-L1 pathway in the treatment of bladder cancer. In the present study, we investigated the prognostic significance of the expression of three genes in the PD-L1 pathway, including PD-L1, B7.1 and PD-1, in three independent bladder cancer datasets in the Gene Expression Omnibus database. PD-L1, B7.1 and PD-1 were significantly associated with clinicopathological parameters indicative of a more aggressive phenotype of bladder cancer, such as a more advanced stage and a higher tumor grade. In addition, a high level expression of PD-L1 was associated with reduced patient survival. Of note, the combination of PD-L1 and B7.1 expression, but not other combinations of the three genes, were also able to predict patient survival. Our findings support the development of anti-PD-L1, which blocks PD-L1-PD-1 and B7.1-PD-L1 interactions, in treatment of bladder cancer. The observations were consistent in the three independent bladder cancer datasets consisting of a total of 695 human bladder specimens. The datasets were then assessed and it was found that the expression levels of the chemokine CC-motif ligand (CCL), CCL3, CCL8 and CCL18, were correlated with the PD-L1 expression level, while ADAMTS13 was differentially expressed in patients with a different survival status (alive or deceased). Additional investigations are required to elucidate the role of these genes in the PD-L1-mediated immune system suppression and bladder cancer progression. In conclusion, findings of this study suggested that PD-L1 is an important prognostic marker and a therapeutic target for bladder cancer.

Immortalization and characterization of human dental mesenchymal cells.

OBJECTIVES: Due to the rarity of human embryonic samples and limited proliferating capability of primary human dental mesenchymal cells, it is valuable to create an immortalized human dental mesenchymal cell line for studying dental mesenchymal cell differentiation and signalling pathways during dentinogenesis in humans. METHODS: In this study, dental mesenchymal cells from human molar tooth germs at 19-week gestation were isolated and immortalized with pSV40. Single cell colonies were then selected by 96-well plate dilution. The immortalized cell line was characterized using immunofluorescent microscopy, RT-PCR and Western blot for the expression of SV40 large T antigen and five genes specific for the mesenchymal stage during tooth development. The differentiation and mineralization activities of the immortalized and primary cells were compared using adipogenic and calcifying induction. RESULTS: The immortalized dental mesenchymal cell line displayed a higher proliferation rate, expressed several tooth-specific markers including Msx1, Pax9, Lhx6, Barx1, and Runx2, and maintained the ability to differentiate and form mineralized nodules. CONCLUSIONS: Our results demonstrated that the immortalized human mesenchymal cell line retained the characteristics similar to primary human dental mesenchymal cells and can be used for studying the mechanisms of human dental mesenchymal cell differentiation and signalling pathways involved in human odontogenesis.

Evaluating DAPK as a therapeutic target.

Death associated protein kinase 1 (DAPK) is an important serine/theoreine kinase involved in various cellular processes such as apoptosis, autophagy and inflammation. DAPK expression and activity are misregulated in multiple diseases including cancer, neuronal death, stoke, et al. Methylation of the DAPK gene is common in many types of cancer and can lead to loss of DAPK expression. In this review, we summarize the pathological status and functional roles of DAPK in disease and compare the published reagents that can manipulate the expression or activity of DAPK. The pleiotropic functions of DAPK make it an intriguing target and the barriers and opportunities for targeting DAPK for future clinical application are discussed.

A Casein kinase 1/Checkpoint kinase 1 pyrazolo-pyridine protein kinase inhibitor as novel activator of the p53 pathway.

Reactivation of the wild-type p53 pathway is one key goal aimed at developing targeted therapeutics in the cancer research field. Although most p53 protein kinases form 'p53-activating' signals, there are few kinases whose action can contribute to the inhibition of p53, as Casein kinase 1 (CK1) and Checkpoint kinase 1 (CHK1). Here we report on a pyrazolo-pyridine analogue showing activity against both CK1 and CHK1 kinases that lead to p53 pathway stabilisation, thus having pharmacological similarities to the p53-activator Nutlin-3. These data demonstrate the emerging potential utility of multivalent kinase inhibitors.