Atovaquone-Proguanil Remains a Potential Stopgap Therapy for Multidrug-Resistant Plasmodium falciparum in Areas along the Thai-Cambodian Border

Our recent report of dihydroartemisinin-piperaquine failure to treat Plasmodium falciparum infections in Cambodia adds new urgency to the search for alternative treatments. Despite dihydroartemisinin-piperaquine failure, and higher piperaquine 50% inhibitory concentrations (IC₅₀s) following reanalysis than those previously reported, P. falciparum remained sensitive to atovaquone (ATQ) in vitro. There were no point mutations in the P. falciparum cytochrome b ATQ resistance gene. Mefloquine, artemisinin, chloroquine, and quinine IC₅₀s remained comparable to those from other recent reports. Atovaquone-proguanil may be a useful stopgap but remains susceptible to developing resistance when used as blood-stage therapy.     

Targeted elimination of breast cancer cells with low proteasome activity is sufficient for tumor regression

Breast cancers are thought to be organized hierarchically with a small number of breast cancer stem cells (BCSCs), able to regrow a tumor after sublethal treatment while their progeny lack this feature. Furthermore, BCSCs are highly resistant to conventional anticancer treatments. According to the cancer stem cell hypothesis, all cancer stem cells in a tumor have to be eliminated to achieve cancer cure. In this study we tested if targeted elimination of BCSCs leads to tumor regression. Specific targeting of BCSCs was achieved via a unique imaging and targeting system that relies on their low proteasome activity. In our system breast cancer cells stably express a fluorescent fusion protein, thymidine kinase-ZsGreen-cODC, which is readily degraded after translation in cells with normal 26S proteasome activity. However, cells with low proteasome activity accumulate this fluorescent fusion protein, thus allowing for their identification, tracking, and specific elimination. Here, we show that the activity of the 26S proteasome was significantly down-regulated in MCF-7, T47D, and MDA-MB-231 cultures enriched for BCSCs. Treatment with ganciclovir resulted in abrogation of sphere formation in vitro, and tumor regression in vivo, thus demonstrating that targeted elimination of BCSCs leads to loss of self-renewal in vitro and tumor regression in vivo. We conclude that specific targeting of BCSCs could be a useful strategy to improve treatment outcome.     

Metabolic differences in breast cancer stem cells and differentiated progeny

In general, tumor cells display a more glycolytic phenotype compared to the corresponding normal tissue. However, it is becoming increasingly clear that tumors are composed of a heterogeneous population of cells. Breast cancers are organized in a hierarchical manner, with the breast cancer stem cells (BCSCs) at the top of the hierarchy. Here, we investigate the metabolic phenotype of BCSCs and their differentiated progeny. In addition, we determine the effect of radiation on the metabolic state of these two cell populations. Luminal, basal, and claudin-low breast cancer cell lines were propagated as mammospheres enriched in BCSCs. Lactate production, glucose consumption, and ATP content were compared with differentiated cultures. A metabolic flux analyzer was used to determine the oxygen consumption, extracellular acidification rates, maximal mitochondria capacity, and mitochondrial proton leak. The effect of radiation treatment of the metabolic phenotype of each cell population was also determined. BCSCs consume more glucose, produce less lactate, and have higher ATP content compared to their differentiated progeny. BCSCs have higher maximum mitochondrial capacity and mitochondrial proton leak compared to their differentiated progeny. Radiation treatment enhances the higher energetic state of the BCSCs, while decreasing mitochondrial proton leak. Our study indicated that breast cancer cells are heterogeneous in their metabolic phenotypes and BCSCs reside in a distinct metabolic state compared to their differentiated progeny. BCSCs display a reliance on oxidative phosphorylation, while the more differentiated progeny displays a more glycolytic phenotype. Radiation treatment affects the metabolic state of BCSCs. We conclude that interfering with the metabolic requirements of BCSCs may prevent radiation-induced reprogramming of breast cancer cells during radiation therapy, thus improving treatment outcome.     

Survival and self-renewing capacity of breast cancer initiating cells during fractionated radiation treatment

Introduction  

Recent data indicate a hierarchical organization of many solid cancers, including breast cancer, with a small number of cancer initiating cells (CICs) that have the ability to self-renew and exhibit multi-lineage potency. We, and others, have demonstrated that CICs in breast cancer and glioma are relatively resistant to ionizing radiation if compared to their non-tumorigenic counterparts. However, the extent of the remaining self-renewing capacity of CICs after fractions of radiation is currently unknown. We hypothesized that CICs, in contrast to their non-tumorigenic counterparts, not only survive fractions of ionizing radiation but also retain the CIC phenotype as defined by operational means.     

Randomized,Double-Blind,Placebo-Controlled Clinical Trial of a Two-Day Regimen of Dihydroartemisinin-Piperaquine for Malaria Prevention Halted for Concern over Prolonged Corrected QT Interval

Dihydroartemisinin-piperaquine, the current first-line drug for uncomplicated malaria caused by Plasmodium falciparum and Plasmodium vivax in Cambodia, was previously shown to be of benefit as malaria chemoprophylaxis when administered as a monthly 3-day regimen. We sought to evaluate the protective efficacy of a compressed monthly 2-day treatment course in the Royal Cambodian Armed Forces. The safety and efficacy of a monthly 2-day dosing regimen of dihydroartemisinin-piperaquine were evaluated in a two-arm, randomized, double-blind, placebo-controlled cohort study with 2:1 treatment allocation. Healthy military volunteers in areas along the Thai-Cambodian border where there is a high risk of malaria were administered two consecutive daily doses of 180 mg dihydroartemisinin and 1,440 mg piperaquine within 30 min to 3 h of a meal once per month for a planned 4-month period with periodic electrocardiographic and pharmacokinetic assessment. The study was halted after only 6 weeks (69 of 231 projected volunteers enrolled) when four volunteers met a prespecified cardiac safety endpoint of QTcF (Fridericia''s formula for correct QT interval) prolongation of >500 ms. The pharmacodynamic effect on the surface electrocardiogram (ECG) peaked approximately 4 h after piperaquine dosing and lasted 4 to 8 h. Unblinded review by the data safety monitoring board revealed mean QTcF prolongation of 46 ms over placebo at the maximum concentration of drug in serum (C_max) on day 2. Given that dihydroartemisinin-piperaquine is one of the few remaining effective antimalarial agents in Cambodia, compressed 2-day treatment courses of dihydroartemisinin-piperaquine are best avoided until the clinical significance of these findings are more thoroughly evaluated. Because ECG monitoring is often unavailable in areas where malaria is endemic, repolarization risk could be mitigated by using conventional 3-day regimens, fasting, and avoidance of repeated dosing or coadministration with other QT-prolonging medications. (This study has been registered at ClinicalTrials.gov under registration no. {"type":"clinical-trial","attrs":{"text":"NCT01624337","term_id":"NCT01624337"}}NCT01624337.)     

Ex Vivo Drug Susceptibility Testing and Molecular Profiling of Clinical Plasmodium falciparum Isolates from Cambodia from 2008 to 2013 Suggest Emerging Piperaquine Resistance

Cambodia''s first-line artemisinin combination therapy, dihydroartemisinin-piperaquine (DHA-PPQ), is no longer sufficiently curative against multidrug-resistant Plasmodium falciparum malaria at some Thai-Cambodian border regions. We report recent (2008 to 2013) drug resistance trends in 753 isolates from northern, western, and southern Cambodia by surveying for ex vivo drug susceptibility and molecular drug resistance markers to guide the selection of an effective alternative to DHA-PPQ. Over the last 3 study years, PPQ susceptibility declined dramatically (geomean 50% inhibitory concentration [IC₅₀] increased from 12.8 to 29.6 nM), while mefloquine (MQ) sensitivity doubled (67.1 to 26 nM) in northern Cambodia. These changes in drug susceptibility were significantly associated with a decreased prevalence of P. falciparum multidrug resistance 1 gene (Pfmdr1) multiple copy isolates and coincided with the timing of replacing artesunate-mefloquine (AS-MQ) with DHA-PPQ as the first-line therapy. Widespread chloroquine resistance was suggested by all isolates being of the P. falciparum chloroquine resistance transporter gene CVIET haplotype. Nearly all isolates collected from the most recent years had P. falciparum kelch13 mutations, indicative of artemisinin resistance. Ex vivo bioassay measurements of antimalarial activity in plasma indicated 20% of patients recently took antimalarials, and their plasma had activity (median of 49.8 nM DHA equivalents) suggestive of substantial in vivo drug pressure. Overall, our findings suggest DHA-PPQ failures are associated with emerging PPQ resistance in a background of artemisinin resistance. The observed connection between drug policy changes and significant reduction in PPQ susceptibility with mitigation of MQ resistance supports reintroduction of AS-MQ, in conjunction with monitoring of the P. falciparum mdr1 copy number, as a stop-gap measure in areas of DHA-PPQ failure.