Assessment of Antitumor and Antiproliferative Efficacy and Detection of Protein-Protein Interactions in Cancer Cells from 3D Tumor Spheroids.

When compared to two-dimensional (2D) cell cultures, 3D spheroids have been considered suitable in vitro models for drug discovery research and other studies of drug activity. Based on different 3D cell culture procedures, we describe procedures we have used to obtain 3D tumor spheroids by both the hanging-drop and ultra-low-attachment plate methods and to analyze the antiproliferative and antitumor efficacy of different chemotherapeutic agents, including a peptidomimetic. We have applied this method to breast and lung cancer cell lines such as BT-474, MCF-7, A549, and Calu-3. We also describe a proximity ligation assay of the cells from the spheroid model to detect protein-protein interactions of EGFR and HER2. © 2022 Wiley Periodicals LLC. Basic Protocol 1: Growth of 3D spheroids using the hanging-drop method Basic Protocol 2: Growth of spheroids using ultra-low-attachment plates Support Protocol 1: Cell viability assay of tumor spheroids Support Protocol 2: Antiproliferative and antitumor study in 3D tumor spheroids Support Protocol 3: Proximity ligation assay on cells derived from 3D spheroids.

Efficacy and safety of esophagectomy via left thoracic approach versus via right thoracic approach for middle and lower thoracic esophageal cancer: a multicenter randomized clinical trial (NST1501).

Background: Left thoracic approach (LTA) has been a favorable selection in surgical treatment for esophageal cancer (EC) patients in China before minimally invasive esophagectomy (MIE) is popular. This study aimed to demonstrate whether right thoracic approach (RTA) is superior to LTA in the surgical treatment of middle and lower thoracic esophageal squamous cell carcinoma (TESCC). Methods: Superiority clinical trial design was used for this multicenter randomized controlled two-parallel group study. Between April 2015 and December 2018, cT1b-3N0-1M0 TESCC patients from 14 centers were recruited and randomized by a central stratified block randomization program into LTA or RTA groups. All enrolled patients were followed up every three months after surgery. The software SPSS 20.0 and R 3.6.2. were used for statistical analysis. Efficacy and safety outcomes, 3-year overall survival (OS) and disease-free survival (DFS) were calculated and compared using the Kaplan-Meier method and the log-rank test. Results: A total of 861 patients without suspected upper mediastinal lymph nodes (umLN) were finally enrolled in the study after 95 ineligible patients were excluded. 833 cases (98.7%) were successfully followed up until June 1, 2020. Esophagectomies were performed via LTA in 453 cases, and via RTA in 408 cases. Compared with the LTA group, the RTA group required longer operating time (274.48±78.92 vs. 205.34±51.47 min, P<0.001); had more complications (33.8% vs. 26.3% P=0.016); harvested more lymph nodes (LNs) (23.61±10.09 vs. 21.92±10.26, P=0.015); achieved a significantly improved OS in stage IIIa patients (67.8% vs. 51.8%, P=0.022). The 3-year OS and DFS were 68.7% and 64.3% in LTA arm versus 71.3% and 63.7% in RTA arm (P=0.20; P=0.96). Conclusions: Esophagectomies via both LTA and RTA can achieve similar outcomes in middle or lower TESCC patients without suspected umLN. RTA is superior to LTA and recommended for the surgical treatment of more advanced stage TESCC due to more complete lymphadenectomy. Trial Registration: ClinicalTrials.gov NCT02448979.

p53 missense mutant G242A subverts natural killer cells in sheltering mouse breast cancer cells against immune rejection.

Cancer cells acquire immunoediting ability to evade immune surveillance and thus escape eradication. It is widely known that mutant proteins encoded from tumor suppressor TP53 exhibit gain-of-function in cancer cells, thereby promoting progression; however, how mutant p53 contributes to the sheltering of cancer cells from host anticancer immunity remains unclear. Herein, we report that murine p53 missense mutation G242A (corresponding to human G245A) suppresses the activation of host natural killer (NK) cells, thereby enabling breast cancer cells to avoid immune assault. We found that serial injection of EMT6 breast cancer cells that carry wild-type (wt) Trp53, like normal fibroblasts, promoted NK activity in mice, while SVTneg2 cells carrying Trp53 G242A+/+ mutation decreased NK cell numbers and increased CD8+ T lymphocyte numbers in spleen. Innate immunity based on NK cells and CD8 T cells was reduced in p53 mutant-carrying transgenic mice (Trp53 R172H/+, corresponding to human R175H/+). Further, upon co-culture with isolated NK cells, EMT6 cells substantively activated NK cells and proliferation thereof, increasing interferon-gamma (IFN-γ) production; however, SVTneg2 cells suppressed NK cell activation. Further mechanistic study elucidated that p53 can modulate expression by cancer cells of Mult-1 and H60a, which are activating and inhibitory ligands for NKG2D receptors of NK cells, respectively, to enhance immune surveillance against cancer. Our findings demonstrate that wt p53 is requisite for NK cell-based immune recognition and elimination of cancerous cells, and perhaps more importantly, that p53 missense mutant presence in cancer cells impairs NK cell-attributable responses, thus veiling cancerous cells from host immunity and enabling cancer progression.

Real-World Survival Outcomes Based on EGFR Mutation Status in Chinese Patients With Lung Adenocarcinoma After Complete Resection: Results From the ICAN Study.

INTRODUCTION: The adjuvant treatment of patients with resected lung adenocarcinoma (LUAD) remains unstandardized. We analyzed the survival outcomes of these patients based on EGFR mutation status and adjuvant chemotherapy treatment. METHODS: This noninterventional real-world study (ICAN) enrolled Chinese patients with resected stages I to III LUAD from April 8, 2010, to December 31, 2010. Tumor EGFR mutation status and 3-year disease-free survival (DFS) were determined. The extension phase provided long-term follow-up with overall survival (OS) as the primary end point. Secondary end points included DFS and prognostic factors of survival. Survival outcomes based on adjuvant chemotherapy treatment, EGFR mutation status, and postoperative stage were analyzed post hoc. RESULTS: Among 568 patients in the ICAN cohort, 472 continued to the extension phase and remained eligible. The 3-year DFS rate was 58.8%. In the extension cohort, 260 patients (55.1%) had EGFR-mutant disease and 207 (43.9%) received adjuvant chemotherapy. At a median follow-up of 109.0 (95% confidence interval [CI]: 106.6-111.4) months, median OS and DFS were 103.3 (95% CI: 101.7-104.9) and 67.4 (95% CI: 49.7-85.2) months, respectively. The 5-year OS and DFS rates were 68.9% (95% CI: 64.3-73.6) and 52.9% (95% CI: 48.2-57.7), respectively. EGFR wild-type disease was a significant independent predictor of worse OS (HR = 1.24, 95% CI: 1.07-1.44, p= 0.004) based on the Cox regression analysis of common factors. Post hoc subgroup analysis revealed that survival outcomes were not significantly different with adjuvant chemotherapy regardless of EGFR mutation status across all postoperative stages. CONCLUSIONS: EGFR mutations are common in operable LUAD, and recurrence and mortality after resection were considerable. Adjuvant chemotherapy did not improve survival outcomes, regardless of EGFR mutation status and postoperative stage.

Genomic signatures define three subtypes of EGFR-mutant stage II-III non-small-cell lung cancer with distinct adjuvant therapy outcomes.

The ADJUVANT study reported the comparative superiority of adjuvant gefitinib over chemotherapy in disease-free survival of resected EGFR-mutant stage II-IIIA non-small cell lung cancer (NSCLC). However, not all patients experienced favorable clinical outcomes with tyrosine kinase inhibitors (TKI), raising the necessity for further biomarker assessment. In this work, by comprehensive genomic profiling of 171 tumor tissues from the ADJUVANT trial, five predictive biomarkers are identified (TP53 exon4/5 mutations, RB1 alterations, and copy number gains of NKX2-1, CDK4, and MYC). Then we integrate them into the Multiple-gene INdex to Evaluate the Relative benefit of Various Adjuvant therapies (MINERVA) score, which categorizes patients into three subgroups with relative disease-free survival and overall survival benefits from either adjuvant gefitinib or chemotherapy (Highly TKI-Preferable, TKI-Preferable, and Chemotherapy-Preferable groups). This study demonstrates that predictive genomic signatures could potentially stratify resected EGFR-mutant NSCLC patients and provide precise guidance towards future personalized adjuvant therapy.

Gefitinib Versus Vinorelbine Plus Cisplatin as Adjuvant Treatment for Stage II-IIIA (N1-N2) EGFR-Mutant NSCLC: Final Overall Survival Analysis of CTONG1104 Phase III Trial.

PURPOSE: ADJUVANT-CTONG1104 (ClinicalTrials.gov identifier: NCT01405079), a randomized phase III trial, showed that adjuvant gefitinib treatment significantly improved disease-free survival (DFS) versus vinorelbine plus cisplatin (VP) in patients with epidermal growth factor receptor (EGFR) mutation-positive resected stage II-IIIA (N1-N2) non-small-cell lung cancer (NSCLC). Here, we report the final overall survival (OS) results. METHODS: From September 2011 to April 2014, 222 patients from 27 sites were randomly assigned 1:1 to adjuvant gefitinib (n = 111) or VP (n = 111). Patients with resected stage II-IIIA (N1-N2) NSCLC and EGFR-activating mutation were enrolled, receiving gefitinib for 24 months or VP every 3 weeks for four cycles. The primary end point was DFS (intention-to-treat [ITT] population). Secondary end points included OS, 3-, 5-year (y) DFS rates, and 5-year OS rate. Post hoc analysis was conducted for subsequent therapy data. RESULTS: Median follow-up was 80.0 months. Median OS (ITT) was 75.5 and 62.8 months with gefitinib and VP, respectively (hazard ratio [HR], 0.92; 95% CI, 0.62 to 1.36; P = .674); respective 5-year OS rates were 53.2% and 51.2% (P = .784). Subsequent therapy was administered upon progression in 68.4% and 73.6% of patients receiving gefitinib and VP, respectively. Subsequent targeted therapy contributed most to OS (HR, 0.23; 95% CI, 0.14 to 0.38) compared with no subsequent therapy. Updated 3y DFS rates were 39.6% and 32. 5% with gefitinib and VP (P = .316) and 5y DFS rates were 22. 6% and 23.2% (P = .928), respectively. CONCLUSION: Adjuvant therapy with gefitinib in patients with early-stage NSCLC and EGFR mutation demonstrated improved DFS over standard of care chemotherapy. Although this DFS advantage did not translate to a significant OS difference, OS with adjuvant gefitinib was one of the longest observed in this patient group compared with historic data.

Gb3-cSrc complex in glycosphingolipid-enriched microdomains contributes to the expression of p53 mutant protein and cancer drug resistance via ß-catenin-activated RNA methylation.

Glucosylceramide synthase (GCS) is a key enzyme catalyzing ceramide glycosylation to generate glucosylceramide (GlcCer), which in turn serves as the precursor for cells to produce glycosphingolipids (GSLs). In cell membranes, GSLs serve as essential components of GSL-enriched microdomains (GEMs) and mediate membrane functions and cell behaviors. Previous studies showed that ceramide glycosylation correlates with upregulated expression of p53 hotspot mutant R273H and cancer drug resistance. Yet, the underlying mechanisms remain elusive. We report herewith that globotriaosylceramide (Gb3) is associated with cSrc kinase in GEMs and plays a crucial role in modulating expression of p53 R273H mutant and drug resistance. Colon cancer cell lines, either WiDr homozygous for missense-mutated TP53 (R273H+/+) or SW48/TP53-Dox bearing heterozygous TP53 mutant (R273H/+), display drug resistance with increased ceramide glycosylation. Inhibition of GCS with Genz-161 (GENZ 667161) resensitized cells to apoptosis in these p53 mutant-carrying cancer cells. Genz-161 effectively inhibited GCS activity, and substantially suppressed the elevated Gb3 levels seen in GEMs of p53-mutant cells exposed to doxorubicin. Complex formation between Gb3 and cSrc in GEMs to activate ß-catenin was detected in both cultured cells and xenograft tumors. Suppression of ceramide glycosylation significantly decreased Gb3-cSrc in GEMs, ß-catenin, and methyltransferase-like 3 for m6A RNA methylation, thus altering pre-mRNA splicing, resulting in upregulated expression of wild-type p53 protein, but not mutants, in cells carrying p53 R273H. Altogether, increased Gb3-cSrc complex in GEMs of membranes in response to anticancer drug induced cell stress promotes expression of p53 mutant proteins and accordant cancer drug resistance.

Quality of life with adjuvant gefitinib versus vinorelbine plus cisplatin in patients with completely resected stage II-IIIA (N1-N2) EGFR-mutant non-small-cell lung cancer: Results from the ADJUVANT (CTONG1104) study.

OBJECTIVES: Health-related quality of life (HRQoL) data complement conventional clinical endpoints when comparing adjuvant gefitinib with chemotherapy in patients with early-stage non-small-cell lung cancer (NSCLC) and epidermal growth factor receptor (EGFR) mutations. This study aimed to assess changes in HRQoL with adjuvant gefitinib vs chemotherapy in this patient group. MATERIALS AND METHODS: In the phase III ADJUVANT trial, patients with completely resected, stage II-IIIA (N1-N2), EGFR-mutant NSCLC were randomized (1:1) to receive either gefitinib for 24 months or vinorelbine plus cisplatin (VP) every 3 weeks for four cycles. HRQoL was assessed as a secondary endpoint using the Functional Assessment of Cancer Therapy-Lung Cancer (FACT-L), Lung Cancer Symptom Scale (LCSS) questionnaires, and Trial Outcome Index (TOI) composite score. HRQoL dynamics, improvements, and time to deterioration were compared between groups. RESULTS: At baseline, 104 of 106, and 80 of 87 patients receiving gefitinib and VP, respectively, completed two questionnaires (FACT-L and LCSS). Baseline scores were balanced between groups. Although HRQoL fluctuated and gradually improved in both groups, longitudinally higher scores were reported with gefitinib than VP (FACT-L, odds ratio 418.16, 95 % confidence interval [CI] 2.75-63509.05, p =  0.019; LCSS, 1.13, 1.04-1.22, p =  0.003; TOI, 88.39, 4.40-1775.05, p =  0.003). Time to deterioration in HRQoL was delayed with gefitinib compared with VP (FACT-L, median 69 vs 6 weeks, hazard ratio 0.62, 95 % CI 0.42-0.90, p =  0.013; LCSS, median 45 vs 6 weeks, 0.63, 0.43-0.93, p =  0.020; TOI, median 164 vs 9 weeks, 0.51, 0.33-0.77, p =  0.001). CONCLUSION: Adjuvant gefitinib is associated with improved HRQoL over VP, supporting its use in patients with stage II-IIIA (N1-N2), EGFR-mutant NSCLC.

Rubusoside-assisted solubilization of poorly soluble C6-Ceramide for a pilot pharmacokinetic study.

Although C6-Ceramide has attracted much attention as a possible tumor suppressor, the delivery of C6-Ceramide is still challenging due to its inherent hydrophobicity and insolubility. In this study we explored the use of a natural compound rubusoside (RUB) as a solubilizer to enhance the solubility of a fluorescence-labeled C6-Ceramide (NBD C6-Ceramide) and to characterize its pharmacokinetics and tissue distribution in an animal model. RUB significantly enhanced the solubility of NBD C6-Ceramide by forming nanomicelles, and efficiently delivered NBD C6-Ceramide in rats by oral and intravenous administration. RUB loaded 1.96 % of NBD C6-Ceramide in the nanomicelles and solubilized it to a concentration of 3.6 mg/mL in water. NBD C6-Ceramide in nanomicelles remained stable in aqueous solutions, allowing intravenous administration without the use of any organic solvents or surfactants. After oral administration, NBD C6-Ceramide rapidly rose to peak plasma concentrations within the first 90 min, distributed to tissues, and remained in vivo for more than 24 h. Tissular levels of NBD C6-Ceramide from high to low were associated with heart, lung, cerebellum, testicle, spleen, liver, kidney, and brain. Altogether, our study demonstrated that RUB-assisted nanomicelles can serve as an efficient and convenient delivery system for short-chain C6-Ceramide and enable in vivo evaluation of potential new cancer treatments.

Ceramide-Rubusoside Nanomicelles, a Potential Therapeutic Approach to Target Cancers Carrying p53 Missense Mutations.

Ceramide (Cer) is an active cellular sphingolipid that can induce apoptosis or proliferation-arrest of cancer cells. Nanoparticle-based delivery offers an effective approach for overcoming bioavailability and biopharmaceutics issues attributable to the pronounced hydrophobicity of Cer. Missense mutations of the protein p53, which have been detected in approximately 42% of cancer cases, not only lose the tumor suppression activity of wild-type p53, but also gain oncogenic functions promoting tumor progression and drug resistance. Our previous works showed that cellular Cer can eradicate cancer cells that carry a p53 deletion-mutation by modulating alternative pre-mRNA splicing, restoring wild-type p53 protein expression. Here, we report that new ceramide-rubusoside (Cer-RUB) nanomicelles considerably enhance Cer in vivo bioavailability and restore p53-dependent tumor suppression in cancer cells carrying a p53 missense mutation. Natural RUB encapsulated short-chain C6-Cer so as to form Cer-RUB nanomicelles (∼32 nm in diameter) that substantially enhanced Cer solubility and its levels in tissues and tumors of mice dosed intraperitoneally. Intriguingly, Cer-RUB nanomicelle treatments restored p53-dependent tumor suppression and sensitivity to cisplatin in OVCAR-3 ovarian cancer cells and xenograft tumors carrying p53 R248Q mutation. Moreover, Cer-RUB nanomicelles showed no signs of significant nonspecific toxicity to noncancerous cells or normal tissues, including bone marrow. Furthermore, Cer-RUB nanomicelles restored p53 phosphorylated protein and downstream function to wild-type levels in p53 R172H/+ transgenic mice. Altogether, this study, for the first time, indicates that natural Cer-RUB nanomicelles offer a feasible approach for efficaciously and safely targeting cancers carrying p53 missense mutations.

Role and Therapeutic Targeting of the PI3K/Akt/mTOR Signaling Pathway in Skin Cancer: A Review of Current Status and Future Trends on Natural and Synthetic Agents Therapy.

The mammalian or mechanistic target of rapamycin (mTOR) and associated phosphatidyl-inositiol 3-kinase (PI3K)/protein kinase B (Akt) pathways regulate cell growth, differentiation, migration, and survival, as well as angiogenesis and metabolism. Dysregulation of these pathways is frequently associated with genetic/epigenetic alterations and predicts poor treatment outcomes in a variety of human cancers including cutaneous malignancies like melanoma and non-melanoma skin cancers. Recently, the enhanced understanding of the molecular and genetic basis of skin dysfunction in patients with skin cancers has provided a strong basis for the development of novel therapeutic strategies for these obdurate groups of skin cancers. This review summarizes recent advances in the roles of PI3K/Akt/mTOR and their targets in the development and progression of a broad spectrum of cutaneous cancers and discusses the current progress in preclinical and clinical studies for the development of PI3K/Akt/mTOR targeted therapies with nutraceuticals and synthetic small molecule inhibitors.

(-)-Oleocanthal Combined with Lapatinib Treatment Synergized against HER-2 Positive Breast Cancer In Vitro and In Vivo.

Dysregulation of epidermal growth factor receptor (EGFR)/human epidermal growth factor-2 (HER2) family is a hallmark of aggressive breast cancer. Small-molecule tyrosine kinase inhibitors are among the most effective cancer targeted treatments. (-)-Oleocanthal (OC) is a naturally occurring phenolic secoiridoid lead from extra-virgin olive oil with documented anti-cancer activities via targeting mesenchymal epithelial transition factor (c-Met). Dysregulation of c-Met promotes aggressiveness to breast cancer-targeted therapies. Lapatinib (LP) is an FDA-approved dual EGFR/HER2 inhibitor for HER2-amplified breast cancer. HER2-Positive tumor cells can escape targeted therapies like LP effects by overexpressing c-Met. Combined OC-LP treatment is hypothesized to be mechanistically synergistic against HER2-overexpressing breast cancer. Combined sub-effective treatments of OC-LP resulted in synergistic anti-proliferative effects against the HER2-positive BT-474 and SK-BR-3 breast cancer cell lines, compared to OC or LP monotherapy. Antibody array and Western blot analysis showed that combined OC-LP treatment significantly inhibited EGFR, HER2, and c-Met receptor activation, as well as multiple downstream signaling proteins, compared to individual OC or LP treatment. OC-LP Combination significantly inhibited invasion and migration of breast cancer cells through reduced activation of focal adhesion kinase (FAK) and paxillin. Combined treatment of OC-10 mg/kg with LP-12.5 mg/kg suppressed more than 90% of BT-474 tumor cells growth in a nude mouse xenograft model, compared to individual OC or LP treatment. Activated c-Met, EGFR, HER2, and protein kinase B (AKT) were significantly suppressed in combination-treated mice tumors, compared to OC or LP monotherapy. This study reveals the OC future potential as combination therapy to sensitize HER2-overexpressing breast cancers and significantly reduce required doses of targeted HER family therapeutics.

The Unique Spatial-Temporal Treatment Failure Patterns of Adjuvant Gefitinib Therapy: A Post Hoc Analysis of the ADJUVANT Trial (CTONG 1104).

INTRODUCTION: Adjuvant gefitinib therapy prolonged disease-free survival in patients with resected early-stage EGFR-mutation positive NSCLC in the ADJUVANT study (CTONG 1104). However, treatment failure patterns after gefitinib therapy are less well characterized. METHODS: Overall, 222 stage N1-N2, EGFR-mutant NSCLC patients received gefitinib or vinorelbine plus cisplatin (VP) treatment. Tumor recurrences or metastases occurring during follow-up were defined as treatment failure; sites and data of first treatment failure were recorded. A post hoc analysis of treatment failure patterns which was estimated by Kaplan-Meier and hazard rate curves in modified intention-to-treat patients was conducted. RESULTS: There were 114 recurrences and 10 deaths before recurrence across 124 progression events. Spatial distribution analysis showed that the first metastasis site was most frequently the central nervous system in the gefitinib group (29 of 106 [27.4%]), extracranial metastases were most frequent in the VP group (32 of 87 [36.8%]). Temporal distribution analysis showed lower tumor recurrence with gefitinib than with VP 0 to 21 months post-surgery. However, recurrence with gefitinib showed a constant rate of increase 12 months post-surgery. The first peak of extracranial metastasis appeared during 9 to 15 months with VP and 24 to 30 months with gefitinib. The highest peak for central nervous system metastases post-surgery occurred after 12 to 18 months with VP and 24 to 36 months with gefitinib. CONCLUSIONS: Adjuvant gefitinib showed advantages over VP chemotherapy in treatment failure patterns especially in extracranial metastasis. Adjuvant tyrosine kinas inhibitors may be considered as a treatment option in resected stage N1-N2 EGFR-mutant NSCLC but longer duration should be explored.

An N6-methyladenosine at the transited codon 273 of p53 pre-mRNA promotes the expression of R273H mutant protein and drug resistance of cancer cells.

Mutant p53 proteins that promote cancer cell invasive growth, metastasis and drug resistance emerge as therapeutic targets. Previously, we reported that suppression of ceramide glycosylation restored wild-type p53 protein and tumor suppressing function in cancer cells heterozygously carrying p53 R273H, a hot-spot missense mutation; however, the mechanisms underlying the control of mutant protein expression remain elusive. Herein, we report that an N6-methyladenosine (m6A) at the point-mutated codon 273 (G > A) of p53 pre-mRNA determines the mutant protein expression. Methylation of the transited adenosine was catalyzed by methyltransferase like 3 (METTL3), and this m6A-RNA promoted a preferential pre-mRNA splicing; consequently, the produced p53 R273H mutant protein resulted in acquired multidrug resistance in colon cancer cells. Furthermore, glycosphingolipids (particularly globotriaosylceramide) generated from serial ceramide glycosylation were seen to activate cSrc and ß-catenin signaling so as to upregulate METTL3 expression, in turn promoting expression of p53 R273H mutant protein, with consequent drug resistance. Conversely, either silencing METTL3 expression by using small interfering RNA (siRNA) or inhibiting RNA methylation with neplanocin A suppressed m6A formation in p53 pre-mRNA, and substantially increased the level of phosphorylated p53 protein (Ser15) and its function in cells heterozygously carrying the R273H mutation, thereby re-sensitizing these cells to anticancer drugs. Concordantly, suppression of ceramide glycosylation repressed METTL3 expression and m6A formation in p53 pre-mRNA, thus sensitizing cells carrying R273H to anticancer drugs. This study uncovers a novel function of pre-mRNA m6A as a determinant of mutant protein expression in cancer cells heterozygously carrying the TP53 R273H mutation. Suppressing both RNA methylation and ceramide glycosylation might constitute an efficacious and specific approach for targeting TP53 missense mutations coding for a G > A transition, thereby improving cancer treatments.

Fluorescence HPLC Analysis of the in-vivo Activity of Glucosylceramide Synthase.

Almost all functions of cells or organs rely on the activities of cellular enzymes. Indeed, the in-vivo activities that directly represent the cellular effects of enzymes in live organs are critical importance to appreciate the roles enzymes play in modulating physiological or pathological processes, although assessments of such in-vivo enzyme activity are more difficult than typical test-tube assays. Recently, we, for the first time, developed a direct and easy-handling method for HPLC analyzing the in-vivo activity of glucosylceramide synthase (GCS). GCS that converts ceramide into glucosylceramide is a limiting-enzyme in the syntheses of glycosphingolipids and is one cause of cancer drug resistance. In our method developed, rubusoside nanomicelles delivers fluorescence N-[6-[(7-nitro-2,1,3-benzoxadiazol-4-yl)amino]hexanoyl]-d-erythro-sphingosine (NBD C6-ceramide) into mice, tissues uptake the cell-permeable substrate, and GCS converts it into NBD C6-glucosylceramide in all organs simultaneously. Further, HPLC analyzes the extracted NBD C6-glucosylceramide to assess alterations of the in-vivo GCS activities in tissues. This method can be broadly used to assess the in-vivo GCS activities in any kind of animal models to appreciate either the role GCS plays in diseases or the therapeutic efficacies of GCS inhibitors.

Graviola (Annona muricata) Exerts Anti-Proliferative, Anti-Clonogenic and Pro-Apoptotic Effects in Human Non-Melanoma Skin Cancer UW-BCC1 and A431 Cells In Vitro: Involvement of Hedgehog Signaling.

Non-melanoma skin cancers (NMSCs) are the leading cause of skin cancer-related morbidity and mortality. Effective strategies are needed to control NMSC occurrence and progression. Non-toxic, plant-derived extracts have been shown to exert multiple anti-cancer effects. Graviola (Annona muricata), a tropical fruit-bearing plant, has been used in traditional medicine against multiple human diseases including cancer. The current study investigated the effects of graviola leaf and stem extract (GLSE) and its solvent-extracted fractions on two human NMSC cell lines, UW-BCC1 and A431. GLSE was found to: (i) dose-dependently suppress UW-BCC1 and A431 cell growth, motility, wound closure, and clonogenicity; (ii) induce G0/G1 cell cycle arrest by downregulating cyclin/cdk factors while upregulating cdk inhibitors, and (iii) induce apoptosis as evidenced by cleavage of caspases-3, -8 and PARP. Further, GLSE suppressed levels of activated hedgehog (Hh) pathway components Smo, Gli 1/2, and Shh while inducing SuFu. GLSE also decreased the expression of pro-apoptotic protein Bax while decreasing the expression of the anti-apoptotic protein Bcl-2. We determined that these activities were concentrated in an acetogenin/alkaloid-rich dichloromethane subfraction of GLSE. Our data identify graviola extracts and their constituents as promising sources for new chemopreventive and therapeutic agent(s) to be further developed for the control of NMSCs.

Gefitinib versus vinorelbine plus cisplatin as adjuvant treatment for stage II-IIIA (N1-N2) EGFR-mutant NSCLC (ADJUVANT/CTONG1104): a randomised, open-label, phase 3 study.

BACKGROUND: Cisplatin-based adjuvant chemotherapy is the standard of care for patients with resected stage II-IIIA non-small-cell lung cancer (NSCLC). RADIANT and SELECT trial data suggest patients with EGFR-mutant stage IB-IIIA resected NSCLC could benefit from adjuvant EGFR tyrosine kinase inhibitor treatment. We aimed to compare the efficacy of adjuvant gefitinib versus vinorelbine plus cisplatin in patients with completely resected EGFR-mutant stage II-IIIA (N1-N2) NSCLC. METHODS: We did a randomised, open-label, phase 3 trial at 27 centres in China. We enrolled patients aged 18-75 years with completely resected (R0), stage II-IIIA (N1-N2), EGFR-mutant (exon 19 deletion or exon 21 Leu858Arg) NSCLC. Patients were stratified by N stage and EGFR mutation status and randomised (1:1) by Pocock and Simon minimisation with a random element to either gefitinib (250 mg once daily) for 24 months or intravenous vinorelbine (25 mg/m2 on days 1 and 8) plus intravenous cisplatin (75 mg/m2 on day 1) every 3 weeks for four cycles. The primary endpoint was disease-free survival in the intention-to-treat population, which comprised all randomised patients; the safety population included all randomised patients who received at least one dose of study medication. Enrolment to the study is closed but survival follow-up is ongoing. The study is registered with ClinicalTrials.gov, number NCT01405079. FINDINGS: Between Sept 19, 2011, and April 24, 2014, 483 patients were screened and 222 patients were randomised, 111 to gefitinib and 111 to vinorelbine plus cisplatin. Median follow-up was 36·5 months (IQR 23·8-44·8). Median disease-free survival was significantly longer with gefitinib (28·7 months [95% CI 24·9-32·5]) than with vinorelbine plus cisplatin (18·0 months [13·6-22·3]; hazard ratio [HR] 0·60, 95% CI 0·42-0·87; p=0·0054). In the safety population, the most commonly reported grade 3 or worse adverse events in the gefitinib group (n=106) were raised alanine aminotransferase and asparate aminotransferase (two [2%] patients with each event vs none with vinorelbine plus cisplatin). In the vinorelbine plus cisplatin group (n=87), the most frequently reported grade 3 or worse adverse events were neutropenia (30 [34%] patients vs none with gefitinib), leucopenia (14 [16%] vs none), and vomiting (eight [9%] vs none). Serious adverse events were reported for seven (7%) patients who received gefitinib and 20 (23%) patients who received vinorelbine plus cisplatin. No interstitial lung disease was noted with gefitinib. No deaths were treatment related. INTERPRETATION: Adjuvant gefitinib led to significantly longer disease-free survival compared with that for vinorelbine plus cisplatin in patients with completely resected stage II-IIIA (N1-N2) EGFR-mutant NSCLC. Based on the superior disease-free survival, reduced toxicity, and improved quality of life, adjuvant gefitinib could be a potential treatment option compared with adjuvant chemotherapy in these patients. However, the duration of benefit with gefitinib after 24 months might be limited and overall survival data are not yet mature. FUNDING: Guangdong Provincial Key Laboratory of Lung Cancer Translational Medicine; National Health and Family Planning Commission of People's Republic of China; Guangzhou Science and Technology Bureau; AstraZeneca China.

A peptidomimetic with a chiral switch is an inhibitor of epidermal growth factor receptor heterodimerization.

Among different types of EGFR dimers, EGFR-HER2 and HER2-HER3 are well known in different types of cancers. Targeting dimerization of EGFR will have a significant impact on cancer therapies. A symmetric peptidomimetic was designed to inhibit the protein-protein interaction of EGFR. The peptidomimetic (Cyclo(1,10)PpR (R) Anapa-FDDF-(R)-Anapa)R, compound 18) was shown to exhibit antiproliferative activity with an IC50 of 194 nM in HER2-expressing breast cancer cell lines and 18 nM in lung cancer cell lines. The peptidomimetic has a Pro-Pro sequence in the structure to stabilize the ß-turn and a ß-amino acid, amino napthyl propionic acid. To investigate the effect of the chirality of ß-amino acid on the structure of the peptide and its antiproliferative activity, diastereoisomers of compound 18 were designed and synthesized. Structure-activity relationships of these compounds indicated that there is a chiral switch at ß-amino acid in the designed compound. The peptidomimetic with R configuration at ß-amino acid and with a L-Pro-D-Pro sequence was the most active compound (18). Using enzyme complement fragmentation assay and proximity ligation assay, we show that compound 18 inhibits HER2:HER3 and EGFR:HER2 dimerization. Surface plasmon resonance studies suggested that compound 18 binds to the HER2 extracellular domain and in particular to domain IV. The anticancer activity of compound 18 was evaluated using a xenograft model of breast cancer in mice; compound 18 suppressed the tumor growth in mice compared to control. Compound 18 was also shown to have a synergistic effect with erlotinib on EGFR mutated lung cancer cell lines.