Complex tumour ecosystem comprising tumour cells and its associated tumour microenvironment (TME) constantly influence the tumoural behaviour and ultimately impact therapy failure, disease progression, recurrence and poor overall survival of patients. Crosstalk between tumour cells and TME amplifies the complexity by creating metabolic changes such as hypoxic environment and nutrient fluctuations. These changes in TME initiate stem cell-like programmes in cancer cells, contribute to tumoural heterogeneity and increase tumour robustness. Recent studies demonstrate the multifaceted role of autophagy in promoting fibroblast production, stemness, cancer cell survival during longer periods of dormancy, eventual growth of metastatic disease and disease resistance. Recent ongoing studies examine autophagy/mitophagy as a powerful survival strategy in response to environmental stress including nutrient deprivation, hypoxia and environmental stress in TME. It prevents irreversible senescence, promotes dormant stem-like state, induces epithelial–mesenchymal transition and increases migratory and invasive potential of tumour cells. The present review discusses various theories and mechanisms behind the autophagy-dependent induction of cancer stem cell (CSC) phenotype. Given the role of autophagic functions in CSC aggressiveness and therapeutic resistance, various mechanisms and studies based on suppressing cellular plasticity by blocking autophagy as a powerful therapeutic strategy to kill tumour cells are discussed.

The term ‘autophagy’ literally translates to ‘self-eating’ and alterations to autophagy have been identified as one of the several molecular changes that occur with aging in a variety of species. Autophagy and aging, have a complicated and multifaceted relationship that has recently come to light thanks to breakthroughs in our understanding of the various substrates of autophagy on tissue homoeostasis. Several studies have been conducted to reveal the relationship between autophagy and age-related diseases. The present review looks at a few new aspects of autophagy and speculates on how they might be connected to both aging and the onset and progression of disease. Additionally, we go over the most recent preclinical data supporting the use of autophagy modulators as age-related illnesses including cancer, cardiovascular and neurodegenerative diseases, and metabolic dysfunction. It is crucial to discover important targets in the autophagy pathway in order to create innovative therapies that effectively target autophagy. Natural products have pharmacological properties that can be therapeutically advantageous for the treatment of several diseases and they also serve as valuable sources of inspiration for the development of possible new small-molecule drugs. Indeed, recent scientific studies have shown that several natural products including alkaloids, terpenoids, steroids, and phenolics, have the ability to alter a number of important autophagic signalling pathways and exert therapeutic effects, thus, a wide range of potential targets in various stages of autophagy have been discovered. In this review, we summarised the naturally occurring active compounds that may control the autophagic signalling pathways.

We report the pharmacological activity of organoruthenium complexes containing chloroquine (CQ) as a chelating ligand. The complexes displayed intraerythrocytic activity against CQ-sensitive 3D7 and CQ-resistant W2 strains of Plasmodium falciparum, with potency and selectivity indexes similar to those of CQ. Complexes displayed activity against all intraerythrocytic stages, but moderate activity against Plasmodium berghei liver stages. However, unlike CQ, organoruthenium complexes impaired gametocyte viability and exhibited fast parasiticidal activity against trophozoites for P. falciparum. This functional property results from the ability of complexes to quickly induce oxidative stress. The parasitaemia of P. berghei-infected mice was reduced by treatment with the complex. Our findings demonstrated that using chloroquine for the synthesis of organoruthenium complexes retains potency and selectivity while leading to an increase in the spectrum of action and parasite killing rate relative to CQ.

Intraerythrocytic malaria parasites ingest the cytosol of their host cell and digest it inside their acid food vacuoles. Acidified (pH 4–5·5, 37 °C) human red blood cell lysates were used to simulate this process, measuring the denaturation of haemoglobin (Hb) and the release of iron, in the absence or presence of exogenous protease. Spontaneous Hb denaturation and appearance of non-heme iron were observed upon lysate acidification, their rates decreasing with increasing pH, and increasing in the presence of protease. Both processes were inhibited by the quinoline-containing anti-malarial drugs (QCDs) chloroquine, quinine, mefloquine and amodiaquine at concentrations well below those expected in the acidic food vacuole of the parasite. Spectrophotometric analysis indicated that chloroquine complexes with heme in acid-denatured haemoglobin. Other weak bases as well as verapamil and diltiazem, known to reverse the resistance of malarial parasites to chloroquine, were without efifect indicating that the action of QCDs is specific. Based on our previous results and the present report, we suggest that iron release in acidified lysates is mediated through the formation of ferryl (Fe(IV)) radicals. QCDs possibly complex with this radical, as they do with heme, and prevent its contact with an adjacent heme molecule which is required for ring opening and iron release. These results may suggest that one of the anti-malarial effects of QCDs is to deprive the parasite of an adequate iron supply. Addition of iron to cultures of Plasmodium falciparum was expected to circumvent the deprivation of iron and reduce the anti-malarial eflfect of QCDs. However, adding iron as penetrating fructose or nitrilotriacetate complexes did not alter the parasite's susceptibility to chloroquine. Ascorbate markedly increased the release of iron in acidified lysates, and this effect was not reduced by chloroquine. Ascorbate was found to decrease parasite susceptibility to chloroquine, suggesting that iron deprivation may be an important factor in the anti-malarial action of QCDs.

In an area of holoendemic malaria in Northern Tanzania, Anopheles gambiae s.l. females were age-graded by Polovodova's method and dissected for sporozoites. Age-specific sporozoite rates implied that mosquitoes acquired new infections at all ages. The extrinsic period lasted just over 3 gonotrophic cycles (9–11 days). Very high sporozoite rates in the oldest females implied the absence or rarity of genetic refractoriness to infection. A method is described for estimating the proportion of bloodmeals which result in mosquito infection. This method makes relatively few assumptions about mosquito behaviour, and could be useful for evaluating transmission-blocking interventions. Overall, it is estimated that about 21% of meals are infectious. This is much higher than previous estimates derived either from experimental mosquito feeding studies or from similar age-grading data collected from the same area in 1962. Various alternative explanations are considered, and it is concluded that there has been a 2.5-fold increase in human infectiousness in the last 25 years. This is partly attributable to suppression of human infectiousness by widespread chloroquine usage during the 1960s, followed by removal of this effect by drug resistance. It is argued that chloroquine would be expected to select for increased infectivity in the parasite, and this may also have contributed to the observed increase.

Reversal of chloroquine (CQ) resistance by verapamil, a Ca2+ antagonist, has been shown in CQ-resistant human and rodent malaria parasites. Here, we report ultrastructural changes associated with this phenomenon in CQ-resistant Plasmodium chabaudi (AS strain) after infected mice were administered CQ and verapamil. At parasitaemias of 5–7%, CQ at 6 mg/kg caused little morphological effect on CQ-resistant parasites. In contrast, co-administration of CQ and verapamil at 50 mg/kg induced swelling of food vacuoles with clumped pigment at 2.5 h. Morphological changes other than food vacuole enlargement occurred at 21 and 45 h: disappearance of endoplasmic reticulum, formation of myelin structures, focal cytoplasmic vacuolization and coarse clumping of electron-dense material in nuclei. These structural changes appeared to be very similar to those observed in CQ-sensitive P. chabaudi in mice injected with CQ alone or CQ plus verapamil. On the other hand, verapamil at 50 mg/kg alone did not induce any effect on both CQ-sensitive and CQ-resistant P. chabaudi. These results suggest that swelling of the food vacuoles is an initial event associated with reversal of CQ-resistance by verapamil.

Chloroquine-resistance in Plasmodium falciparum is associated with polymorphisms in a locus on or near the cg2 gene on chromosome 7, and in the pfmdr1 gene on chromosome 5. In this study we typed P. falciparum DNA from uncomplicated malaria cases in The Gambia in 1990, 1995 and 1996 for size polymorphism in the omega repeat of cg2, for sequence polymorphisms in pfmdr1 at codons 86 and 184, in dhfr at codon 108 and in the msp2 gene. Chloroquine sensitivity tests were conducted in vitro. A significant but incomplete association was found between the presence of the cg2 Dd2-like omega repeat size polymorphism and in vitro resistance, and between the tyr-86 allele of pfmdr1 and in vitro resistance. Furthermore there was strong linkage disequilibrium between the pfmdr1 asn-86 allele and the cg2 not Dd2-like omega repeat allele located on different chromosomes. In contrast, no linkage disequilibrium was found between these alleles and either the dhfr ser-108 allele or the msp2 IC sequence polymorphism. No significant linkage was measured between pfmdr1 asn-86 and phe-184 although these loci are separated only by 296 base pairs. Our results suggest that genetic elements linked to the cg2 and the pfmdr1 genes are important determinants of chloroquine resistance. It can be concluded that the observed linkage disequilibrium is maintained epistatically through selection by chloroquine.

Malaria parasites are capable of modulating the diversion of resources from asexual growth to the production of stages infective to mosquitoes (gametocytes). Increased rates of gametocytogenesis appear to be a general response to stress, both naturally encountered and novel. We have previously reported earlier and greater gametocytogenesis in response to subcurative antimalarial chemotherapy in the rodent malaria, Plasmodium chabaudi, in vivo. Using an immunofluorescent assay to detect parasites that had invaded red blood cell monolayers, we demonstrate a 5-fold increase in gametocytogenesis in the human malaria, P. falciparum, in vitro, in response to treatment with the antimalarial drug chloroquine. In all clones used, gametocytogenesis increased with increasing inhibition of asexual growth by chloroquine. Furthermore, there were clone differences in the relationship between stress and gametocyte production, implying the response was genetically variable. This was not, however, associated with chloroquine resistance. The epidemiological significance of these results is discussed.

We have attempted to provide a rational basis for improving the protocols for chemotherapy of malaria. We model the regression of parasitaemia by Plasmodium falciparum, its subsequent elimination from the body, or recrudescence, for populations of cells treated with chloroquine. Our model assumes that the drug forms a complex with some receptor in the parasite and that parasites possessing this complex die at a defined rate. We take into account that chloroquine is eliminated exponentially from the body. We show how the parameters of the model can be derived from observations in the field. The model correctly predicts the effects of drug dose, degree of initial parasitaemia, rate of parasite multiplication and degree of drug resistance to chloroquine chemotherapy. The level of parasitaemia will reduce to a minimum at sufficiently high concentrations of chloroquine, but only if the parasitaemia is reduced to below that of 1 parasite per infected person will a cure of malaria be obtained. Otherwise, recrudescence will, sooner or later, occur. We show that, even for drug-resistant malaria, if 2 doses of chloroquine are given to a patient with an interval of some 10 days between them, parasites can be eliminated from the body without toxic levels of chloroquine being reached.

Despite considerable therapeutic success with the antimalarial 4-aminoquinolines such as chloroquine, there is serious doubt about the future of this drug class due mainly to the development and spread of parasite resistance throughout endemic areas. In this article we review the possible biochemical and molecular basis of resistance. Based on our current understanding we have considered the possibility of developing strategies which may allow the aminoquinolines to once again be used effectively against P. falciparum. Our conclusions are that drug resistance is the result of a reduced rate of drug uptake which in turn reduces the amount of drug available to bind the target. The basis for this reduced accumulation could be an altered pH gradient making the food vacuole more alkaline or the parasite cytosol more acidic, an efflux pump removing drug directly from the membrane or any other process which will reduce the rate of drug uptake. Central to the effectiveness of this resistance mechanism is the transient availability of a high affinity, low capacity drug binding site (possibly haem) within the parasite. Resistance reversers such as verapamil influence the apparent Ka for this drug binding phenomenon via an increased drug uptake rate. We demonstrate that by chemical modification of the aminoquinolines, producing predictable alterations in their physicochemical properties, that it is possible to minimise the verapamil sensitive component of resistance and reduce significantly cross-resistance patterns without loss in absolute activity. Based on these views we suggest that the aminoquinoline antimalarials still have a role to play in the cheap, safe and effective chemotherapy of falciparum malaria.

The 4-aminoquinolines chloroquine (CQ) and amodiaquine (AM) were used to treat Gambian children with uncomplicated falciparum malaria in a randomized drug trial. Blood samples were taken immediately before treatment (day 0), and at day 7 and day 28 after treatment. Samples from those parasitologically positive at day 7 following treatment (‘early positives’) and those positive at day 28 but negative at day 7 (‘late positives’) have been studied by PCR followed by restriction enzyme digestion to determine the allelic status of the pfmdr 1 locus at the codon-86 position (asparagine or tyrosine), previously associated with resistance to CQ. A significantly higher prevalence of the tyr-86 allele was observed in samples taken immediately before treatment (day 0) in the early positives group when compared with the late positives group. This suggests the tyr-86 allele contributes to drug resistance in the early positives group. This association remained significant for both CQ and AM groups, implying a common genetic basis of resistance. Predominance of the allele at day 7 is consistent with a strong selection in the first week following treatment. In the late positives group, a significantly higher prevalence of the tyr-86 allele was observed in the samples at day 28 when compared with those at day 0, suggestive of selection during the period day 7 to day 28. Differences were observed in the extent of this selection in the CQ and AM groups. The samples were genotyped at 3 unlinked polymorphic loci. These analyses suggested that most parasites observed at day 7 were probably recrudescences whereas most of those at day 28 were reinfections.