Shikonin Inhibits Cell Growth of Sunitinib-Resistant Renal Cell Carcinoma by Activating the Necrosome Complex and Inhibiting the AKT/mTOR Signaling Pathway.

Therapy resistance remains a major challenge in treating advanced renal cell carcinoma (RCC), making more effective treatment strategies crucial. Shikonin (SHI) from traditional Chinese medicine has exhibited antitumor properties in several tumor entities. We, therefore, currently investigated SHI's impact on progressive growth and metastatic behavior in therapy-sensitive (parental) and therapy-resistant Caki-1, 786-O, KTCTL-26, and A498 RCC cells. Tumor cell growth, proliferation, clonogenic capacity, cell cycle phase distribution, induction of cell death (apoptosis and necroptosis), and the expression and activity of regulating and signaling proteins were evaluated. Moreover, the adhesion and chemotactic activity of the RCC cells after exposure to SHI were investigated. SHI significantly inhibited the growth, proliferation, and clone formation in parental and sunitinib-resistant RCC cells by G2/M phase arrest through down-regulation of cell cycle activating proteins. Furthermore, SHI induced apoptosis and necroptosis by activating necrosome complex proteins. Concomitantly, SHI impaired the AKT/mTOR pathway. Adhesion and motility were cell line specifically affected by SHI. Thus, SHI may hold promise as an additive option in treating patients with advanced and therapy-resistant RCC.

Artesunate Inhibits the Growth Behavior of Docetaxel-Resistant Prostate Cancer Cells.

Novel therapeutic strategies are urgently needed for advanced metastatic prostate cancer (PCa). Phytochemicals used in Traditional Chinese Medicine seem to exhibit tumor suppressive properties. Therefore, the therapeutic potential of artesunate (ART) on the progressive growth of therapy-sensitive (parental) and docetaxel (DX)-resistant PCa cells was investigated. Parental and DX-resistant PCa cell lines DU145, PC3, and LNCaP were incubated with artesunate (ART) [1-100 µM]. ART-untreated and 'non-cancerous' cells served as controls. Cell growth, proliferation, cell cycle progression, cell death and the expression of involved proteins were evaluated. ART, dose- and time-dependently, significantly restricted cell growth and proliferation of parental and DX-resistant PCa cells, but not of 'normal, non-cancerous' cells. ART-induced growth and proliferation inhibition was accompanied by G0/G1 phase arrest and down-regulation of cell cycle activating proteins in all DX-resistant PCa cells and parental LNCaP. In the parental and DX-resistant PC3 and LNCaP cell lines, ART also promoted apoptotic cell death. Ferroptosis was exclusively induced by ART in parental and DX-resistant DU145 cells by increasing reactive oxygen species (ROS). The anti-cancer activity displayed by ART took effect in all three PCa cell lines, but through different mechanisms of action. Thus, in advanced PCa, ART may hold promise as a complementary treatment together with conventional therapy.

Shikonin Reduces Growth of Docetaxel-Resistant Prostate Cancer Cells Mainly through Necroptosis.

The prognosis for advanced prostate carcinoma (PCa) remains poor due to development of therapy resistance, and new treatment options are needed. Shikonin (SHI) from Traditional Chinese Medicine has induced antitumor effects in diverse tumor entities, but data related to PCa are scarce. Therefore, the parental (=sensitive) and docetaxel (DX)-resistant PCa cell lines, PC3, DU145, LNCaP, and 22Rv1 were exposed to SHI [0.1-1.5 µM], and tumor cell growth, proliferation, cell cycling, cell death (apoptosis, necrosis, and necroptosis), and metabolic activity were evaluated. Correspondingly, the expression of regulating proteins was assessed. Exposure to SHI time- and dose-dependently inhibited tumor cell growth and proliferation in parental and DX-resistant PCa cells, accompanied by cell cycle arrest in the G2/M or S phase and modulation of cell cycle regulating proteins. SHI induced apoptosis and more dominantly necroptosis in both parental and DX-resistant PCa cells. This was shown by enhanced pRIP1 and pRIP3 expression and returned growth if applying the necroptosis inhibitor necrostatin-1. No SHI-induced alteration in metabolic activity of the PCa cells was detected. The significant antitumor effects induced by SHI to parental and DX-resistant PCa cells make the addition of SHI to standard therapy a promising treatment strategy for patients with advanced PCa.

Artesunate Impairs Growth in Cisplatin-Resistant Bladder Cancer Cells by Cell Cycle Arrest, Apoptosis and Autophagy Induction.

Cisplatin, which induces DNA damage, is standard chemotherapy for advanced bladder cancer (BCa). However, efficacy is limited due to resistance development. Since artesunate (ART), a derivative of artemisinin originating from Traditional Chinese Medicine, has been shown to exhibit anti-tumor activity, and to inhibit DNA damage repair, the impact of artesunate on cisplatin-resistant BCa was evaluated. Cisplatin-sensitive (parental) and cisplatin-resistant BCa cells, RT4, RT112, T24, and TCCSup, were treated with ART (1-100 µM). Cell growth, proliferation, and cell cycle phases were investigated, as were apoptosis, necrosis, ferroptosis, autophagy, metabolic activity, and protein expression. Exposure to ART induced a time- and dose-dependent significant inhibition of tumor cell growth and proliferation of parental and cisplatin-resistant BCa cells. This inhibition was accompanied by a G0/G1 phase arrest and modulation of cell cycle regulating proteins. ART induced apoptos is by enhancing DNA damage, especially in the resistant cells. ART did not induce ferroptosis, but led to a disturbance of mitochondrial respiration and ATP generation. This impairment correlated with autophagy accompanied by a decrease in LC3B-I and an increase in LC3B-II. Since ART significantly inhibits proliferative and metabolic aspects of cisplatin-sensitive and cisplatin-resistant BCa cells, it may hold potential in treating advanced and therapy-resistant BCa.

Artesunate Inhibits Growth of Sunitinib-Resistant Renal Cell Carcinoma Cells through Cell Cycle Arrest and Induction of Ferroptosis.

Although innovative therapeutic concepts have led to better treatment of advanced renal cell carcinoma (RCC), efficacy is still limited due to the tumor developing resistance to applied drugs. Artesunate (ART) has demonstrated anti-tumor effects in different tumor entities. This study was designed to investigate the impact of ART (1-100 µM) on the sunitinib-resistant RCC cell lines, Caki-1, 786-O, KTCTL26, and A-498. Therapy-sensitive (parental) and untreated cells served as controls. ART's impact on tumor cell growth, proliferation, clonogenic growth, apoptosis, necrosis, ferroptosis, and metabolic activity was evaluated. Cell cycle distribution, the expression of cell cycle regulating proteins, p53, and the occurrence of reactive oxygen species (ROS) were investigated. ART significantly increased cytotoxicity and inhibited proliferation and clonogenic growth in both parental and sunitinib-resistant RCC cells. In Caki-1, 786-O, and A-498 cell lines growth inhibition was associated with G0/G1 phase arrest and distinct modulation of cell cycle regulating proteins. KTCTL-26 cells were mainly affected by ART through ROS generation, ferroptosis, and decreased metabolism. p53 exclusively appeared in the KTCTL-26 cells, indicating that p53 might be predictive for ART-dependent ferroptosis. Thus, ART may hold promise for treating selected patients with advanced and even therapy-resistant RCC.

Amygdalin Modulates Prostate Cancer Cell Adhesion and Migration In Vitro.

The natural compound, amygdalin, is notably popular with prostate cancer patients as an alternative or complementary treatment option. However, knowledge about its mode of action is sparse. We investigated amygdalin's impact on prostate cancer adhesion and motile behavior. DU-145 and PC3 cancer cells were exposed to amygdalin. Adhesion to human vascular endothelium or immobilized collagen was then explored. The influence of amygdalin on chemotaxis and migration was also investigated, as well as amygdalin induced alteration to surface and total cellular α and ß integrin expression. Integrin knockdown was performed to evaluate the integrin influence on chemotaxis and adhesion. Amygdalin significantly reduced chemotactic activity, migration, and adhesion of DU-145 but not of PC3 cells. Amygdalin elevated integrin α2 in both cell lines. Integrin α6 was reduced by amygdalin only in DU-145 cells, whereas ß1 increased only in PC3 cells. Functional blocking revealed a negative association of α2 with PC3 and DU-145 chemotaxis. The ß1 increase correlated with enhanced chemotaxis, the diminished α6 expression with reduced chemotaxis. Amygdalin acted on prostate cancer cells in vitro. It induced downregulation of α6 integrin in DU-145 but not in PC3 cells, suggesting that exposing certain prostate cancer cells to amygdalin might inhibit metastatic spread promoted by this particular integrin.

Cyanide and lactate levels in patients during chronic oral amygdalin intake followed by intravenous amygdalin administration.

The natural compound amygdalin has gained high popularity among tumor patients as a complementary or alternative treatment option. However, due to metabolization of amygdalin to cyanide (HCN) following oral consumption, there could be a high risk of lactic acidosis caused by cyanide intoxication. The present retrospective study was undertaken to evaluate cyanide blood and lactate plasma levels of tumor patients (n = 55) before and after intravenous (i.v.) amygdalin infusion. All patients had also continuously ingested amygdalin tablets (3 x 500 mg/day), excepting on the days of i.v. administration. Each patient received one to five intravenous amygdalin treatments. The time period between each i.v. application ranged between 4-6 days. The initial i.v. dose was 6 mg (n = 28), 9 mg (n = 1), 15 mg (n = 1) or 18 mg (n = 25). The mean cyanide blood level before i.v. amygdalin administration was 34.74 µg/L, which increased significantly to a mean value of 66.20 µg/L after i. v. amygdalin application. In contrast, lactate decreased significantly from 1266 µmol/L pre-infusion to 868 µmol/L post-infusion. Increasing i.v. amygdalin by 1 mg was also associated with a significant increase in the cyanide level, while the lactate blood level significantly decreased. This is the first study evaluating cyanide levels under conditions employed by amygdalin administrators, i.e. after chronic oral amygdalin intake and then again after a closely subsequent intravenous amygdalin administration. Since lactate decreased, whilst cyanide increased, it is concluded that elevation of cyanide does not induce metabolic acidosis in terms of an increased lactate level.

The Antitumor Effect of Curcumin in Urothelial Cancer Cells Is Enhanced by Light Exposure In Vitro.

The natural compound curcumin exerts antitumor properties in vitro, but its clinical application is limited due to low bioavailability. Light exposure in skin and skin cancer cells has been shown to improve curcumin bioavailability; thus, the object of this investigation was to determine whether light exposure might also enhance curcumin efficacy in bladder cancer cell lines. RT112, UMUC3, and TCCSUP cells were preincubated with low curcumin concentrations (0.1-0.4 µg/ml) and then exposed to 1.65 J/cm2 visible light for 5 min. Cell growth, cell proliferation, apoptosis, cell cycle progression, and cell cycle regulating proteins along with acetylation of histone H3 and H4 were investigated. Though curcumin alone did not alter cell proliferation or apoptosis, tumor cell growth and proliferation were strongly blocked when curcumin was combined with visible light. Curcumin-light caused the bladder cancer cells to become arrested in different cell phases: G0/G1 for RT112, G2/M for TCCSUP, and G2/M- and S-phase for UMUC3. Proteins of the Cdk-cyclin axis were diminished in RT112 after application of 0.1 and 0.4 µg/ml curcumin. Cell cycling proteins were upregulated in TCCSUP and UMUC3 in the presence of 0.1 µg/ml curcumin-light but were partially downregulated with 0.4 µg/ml curcumin. 0.4 µg/ml (but not 0.1 µg/ml) curcumin-light also evoked late apoptosis in TCCSUP and UMUC3 cells. H3 and H4 acetylation was found in UMUC3 cells treated with 0.4 µg/ml curcumin alone or with 0.1 µg/ml curcumin-light, pointing to an epigenetic mechanism. Light exposure enhanced the antitumor potential of curcumin on bladder cancer cells but by different molecular action modes in the different cell lines. Further studies are necessary to evaluate whether intravesical curcumin application, combined with visible light, might become an innovative tool in combating bladder cancer.

Sulforaphane as an adjunctive to everolimus counteracts everolimus resistance in renal cancer cell lines.

BACKGROUND: The mechanistic target of rapamycin (mTOR) inhibitors, everolimus and temsirolimus, have widened therapeutic options to treat renal cell carcinoma (RCC). However, chronic treatment with these inhibitors often induces resistance, leading to therapeutic failure. PURPOSE: The natural compound, sulforaphane (SFN), was added to an everolimus based regime in vitro in the hopes of preventing resistance development. METHODS: A panel of RCC cell lines (A498, Caki-1, KTCTL-26) was treated with everolimus or SFN or with an everolimus-SFN-combination, either short- (24h) or long-term (8 weeks), and cell growth, proliferation, apoptosis, and cell cycle phases were measured. The cell cycle regulating proteins cdk1, cdk2, cyclin A, cyclin B, akt and raptor (both total and activated) were also evaluated. RESULTS: Short-term incubation with everolimus (1nM) or SFN (5µM) significantly reduced RCC cell growth. Additive effects on tumor growth and proliferation were evoked by the SFN-everolimus combination. Long-term everolimus-incubation led to resistance development in Caki-1 cells, evidenced by elevated growth and proliferation, associated with an increased percentage of G2/M (non-synchronized cell model) or S-phase (synchronized cell model) cells. Molecular analysis revealed up-regulation of the cdk1-cyclin B and cdk2-cyclin A axis, along with elevated phosphorylation of the mTOR sub-member, raptor. In contrast, resistance development was not observed with the long-term combination of SFN-everolimus. The combination suppressed Caki-1 growth and proliferation, and was associated with an increase in G0/G1-phase cells, diminished cdk1 and akt (both total and activated), cyclin B and raptor expression. CONCLUSION: Adding SFN to an everolimus based RCC treatment regimen in vitro delayed resistance development observed with chronic everolimus monotherapy. Ongoing in vivo studies are necessary to verify the in vitro data.

Sulforaphane inhibits proliferation and invasive activity of everolimus-resistant kidney cancer cells in vitro.

Although the mechanistic target of rapamycin (mTOR) inhibitor, everolimus, has improved the outcome of patients with renal cell carcinoma (RCC), improvement is temporary due to the development of drug resistance. Since many patients encountering resistance turn to alternative/complementary treatment options, an investigation was initiated to evaluate whether the natural compound, sulforaphane (SFN), influences growth and invasive activity of everolimus-resistant (RCCres) compared to everolimus-sensitive (RCCpar) RCC cell lines in vitro. RCC cells were exposed to different concentrations of SFN and cell growth, cell proliferation, apoptosis, cell cycle, cell cycle regulating proteins, the mTOR-akt signaling axis, adhesion to human vascular endothelium and immobilized collagen, chemotactic activity, and influence on surface integrin receptor expression were investigated. SFN caused a significant reduction in both RCCres and RCCpar cell growth and proliferation, which correlated with an elevation in G2/M- and S-phase cells. SFN induced a marked decrease in the cell cycle activating proteins cdk1 and cyclin B and siRNA knock-down of cdk1 and cyclin B resulted in significantly diminished RCC cell growth. SFN also modulated adhesion and chemotaxis, which was associated with reduced expression of the integrin subtypes α5, α6, and ß4. Distinct differences were seen in RCCres adhesion and chemotaxis (diminished by SFN) and RCCpar adhesion (enhanced by SFN) and chemotaxis (not influenced by SFN). Functional blocking of integrin subtypes demonstrated divergent action on RCC binding and invasion, depending on RCC cell sensitivity to everolimus. Therefore, SFN administration could hold potential for treating RCC patients with established resistance towards everolimus.

Evaluation of TKTL1 as a biomarker in serum of prostate cancer patients.

INTRODUCTION: Monocyte associated transketolase-like 1 (TKTL1) as a cancer biomarker has become popular with alternative practitioners, but plays no role in conventional medicine. This investigation evaluates the potential of serum TKTL1 as a biomarker for prostate cancer. MATERIAL AND METHODS: Patients (n = 66) undergoing curative radical prostatectomy (RPE) for biopsy-pro-ven PCa were included in the study. Controls (n = 10) were healthy, age-matched, male volunteers. 10 ml of peripheral blood was drawn from patients several days before surgery and from controls. Serum TKTL1 was measured using the ELISA method. RESULTS: The median age at tumor diagnosis was 66 years and median serum PSA was 8.0 ng/ml. Nearly 96% of PCas submitted to surgery were clinically significant. Compared to healthy controls, serum TKTL1 was significantly lower in PCa patients (p = 0.0001, effect size indicator r = Z/sqr(n) = 0.4179). No correlation was apparent between serum TKTL1 and serum PSA, Gleason sum, tumor stage or further clinical and pathologic parameters. CONCLUSIONS: Reduced serum TKTL1 in PCa patients stands in opposition to TKTL1 epitope detection in monocytes (EDIM) based studies, whereby increased TKTL1 in monocytes of tumor patients has been reported. Since serum TKTL1 does not correlate with clinical parameters in the current investigation, further research is needed to clarify whether serum TKTL1 has potential as a biomarker for PCa.

Amygdalin, quackery or cure?

BACKGROUND: The cyanogenic diglucoside, amygdalin, has gained high popularity among cancer patients together with, or in place of, conventional therapy. Still, evidence based research on amygdalin is sparse and its benefit controversial. PURPOSE: Since so many cancer patients consume amygdalin, and many clinicians administer it without clear knowledge of its mode of action, current knowledge has been summarized and the pros and cons of its use weighed. METHODS: A retrospective analysis was conducted for amygdalin relevant reports using the PubMed database with the main search term "Amygdalin" or "laetrile", at times combined with "cancer", "patient", "cyanide" or "toxic". We did not exclude any "unwanted" articles. Additionally, internet sources authorized by governmental or national institutions have also been included. SECTIONS: Individual chapters summarize pharmacokinetics, preclinical and clinical studies and toxicity. CONCLUSION: No convincing evidence showing that amygdalin induces rapid, distinct tumor regression in cancer patients, particularly in those with late-stage disease, is apparent. However, there is also no evidence that purified amygdalin, administered in "therapeutic" dosage, causes toxicity. Multiple aspects of amygdalin administration have not yet been adequately explored, making further investigation necessary to evaluate its actual therapeutic potential.

Amygdalin delays cell cycle progression and blocks growth of prostate cancer cells in vitro.

AIMS: Despite impressive survival benefits from new agents to treat metastasized prostate cancer (PCa), progressive drug resistance hinders long-term response and restricts the efficacy of subsequent therapy. Due to reported antitumor activity of amygdalin and growing popularity for complementary and alternative medicine the potential of this natural, widely used substance to exert antineoplastic effects on prostate cancer cells has been assessed. MAIN METHODS: LNCaP (castration-sensitive), DU-145 and PC3 cells (castration-resistant) were exposed to different concentrations of amygdalin for 24h or 2weeks. Cell growth was measured by the MTT test, clonal formation by the clonogenic assay. Flow cytometry served to investigate apoptosis and cell cycle phases. Cell cycle regulating proteins and the mTOR-akt signaling axis were analyzed by western blotting. KEY FINDINGS: Amygdalin dose-dependently diminished tumor cell growth with maximum effects at 10mg/ml. Apoptosis of PC3 and LNCaP but not of DU-145 cells was reduced, whereas colony formation was suppressed in all cell lines. A decrease in the number of G2/M- and S-phase cells along with an elevated number of G0/G1-phase cells was recorded. The cell cycle proteins cdk 1, cdk 2 and cdk 4 as well as cyclin A, cyclin B and cyclin D3 were modulated by amygdalin after both 24h and 2weeks. Distinct effects on p19 and p27 expression and on Akt, Rictor and Raptor activation became evident only after 2weeks. SIGNIFICANCE: Amygdalin exhibits significant antitumor activity in both castration-sensitive and castration-resistant PCa cell lines and merits further evaluation for therapeutic purposes.

Use of complementary and alternative medicine before and after organ removal due to urologic cancer.

OBJECTIVE: Many patients use complementary and alternative medicine (CAM) as primary treatment or symptom relief for a variety of illnesses. This study was designed to investigate the influence of surgical removal of a tumor-bearing urogenital organ on CAM use. METHODS: From 2007 to 2011, 350 patients underwent major urological surgery for kidney, prostate, or bladder cancer at the Goethe-University Hospital, Frankfurt, Germany. Data from 172 patients (49%), who returned a questionnaire, were retrospectively evaluated using the hospital information system along with the questionnaire to objectify CAM use 2 years before and after surgery. RESULTS: From the 172 patients returning questionnaires, 56 (33%) used CAM before and/or after surgery and 116 (67%) never used CAM. Of the 56 CAM users, 30 (54%) used CAM presurgery and 53 (95%) used CAM postsurgery, indicating a significant change of mind about CAM use. Patients of German nationality used CAM significantly more than patients of other nationalities. Higher educational status (high-school diploma or higher) was a significant factor in favor of CAM use. The most common type of CAM used before/after surgery was an alternative medical system (63/49%), a manipulative and body-based method (50/19%), and a biological-based therapy (37/32%). Information about CAM, either provided by medical professionals or by other sources, was the main reason determining whether patients used CAM or not. CONCLUSION: The number of patients using CAM almost doubled after surgical removal of a cancer-bearing organ. Better awareness and understanding of CAM use by medical professionals could improve patient counseling.

Molecular analysis of sunitinib resistant renal cell carcinoma cells after sequential treatment with RAD001 (everolimus) or sorafenib.

Sequential application of target drugs is standard procedure after renal cell carcinoma (RCC) patients develop resistance. To optimize the sequence, antitumour effects of the mTOR inhibitor RAD001 or the tyrosine kinase inhibitor (TKI) sorafenib on RCC cells with acquired resistance to the TKI sunitinib was evaluated. RCC cells were exposed to 1 µM sunitinib for 24 hrs (as control) and for 8 weeks (to induce resistance) and then switched to RAD001 (5 nM) or sorafenib (5 µM) for a further 8 weeks. Tumour cell growth, cell cycle progression, cell cycle regulating proteins and intracellular signalling were then investigated. Short-term application of sunitinib (24 hrs) induced cell growth blockade with accumulation in the G2/M phase. RCC cells became resistant to sunitinib after 8 weeks, demonstrated by accelerated cell growth along with enhanced cdk1, cdk2, loss of p27, activation of Akt, Rictor and Raptor. Switching to sorafenib only slightly reduced growth of the sunitinib resistant RCC cells and molecular analysis indicated distinct cross-resistance. In contrast, full response was achieved when the cancer cells were treated with RAD001. p19 and p27 strongly increased, phosphorylated Akt, Rictor and Raptor decreased and the tumour cells accumulated in G0/G1. It is concluded that an mTOR-inhibitor for second-line therapy could be the strategy of choice after first-line sunitinib failure.