Cyril Rauch

Endocytosis Switch Controlled by Transmembrane Osmotic Pressure and Phospholipid Number Asymmetry

The dynamics of endocytosis in living K562 cells was investigated after the osmotic pressure of the external medium was decreased and the transmembrane phospholipid number asymmetry was increased. When the external pressure was decreased by a factor of 0.54, a sudden inhibition of endocytosis was observed. Under these conditions, the endocytosis suddenly recovered after the phospholipid number asymmetry was increased. The phospholipid asymmetry was generated by the addition of exogenous phosphatidylserine, which is translocated by the endogenous flippase activity to the inner layer of the membrane. The recovery of endocytosis is thus consistent with the view that the phospholipid number asymmetry can act as a budding force for endocytosis. Moreover, we quantitatively predict both the inhibition and recovery of endocytosis as first-order phase transitions, using a general model that assumes the existence of a transmembrane surface tension asymmetry as the budding driving force. In this model, the tension asymmetry is considered to be elastically generated by the activity of phospholipid pumping. We finally propose that cells may trigger genetic transcription responses after the internalization of cytokine-receptor complexes, which could be controlled by variations in the cytosolic or external pressure.

Cariporide and other new and powerful NHE1 inhibitors as potentially selective anticancer drugs--an integral molecular/biochemical/metabolic/clinical approach after one hundred years of cancer research.

In recent years an increasing number of publications have emphasized the growing importance of hydrogen ion dynamics in modern cancer research, from etiopathogenesis and treatment. A proton [H+]-related mechanism underlying the initiation and progression of the neoplastic process has been recently described by different research groups as a new paradigm in which all cancer cells and tissues, regardless of their origin and genetic background, have a pivotal energetic and homeostatic disturbance of their metabolism that is completely different from all normal tissues: an aberrant regulation of hydrogen ion dynamics leading to a reversal of the pH gradient in cancer cells and tissues (↑pHi/↓pHe, or “proton reversal”). Tumor cells survive their hostile microenvironment due to membrane-bound proton pumps and transporters, and their main defensive strategy is to never allow internal acidification because that could lead to their death through apoptosis. In this context, one of the primary and best studied regulators of both pHi and pHe in tumors is the Na+/H+ exchanger isoform 1 (NHE1). An elevated NHE1 activity can be correlated with both an increase in cell pH and a decrease in the extracellular pH of tumors, and such proton reversal is associated with the origin, local growth, activation and further progression of the metastatic process. Consequently, NHE1 pharmaceutical inhibition by new and potent NHE1 inhibitors represents a potential and highly selective target in anticancer therapy. Cariporide, being one of the better studied specific and powerful NHE1 inhibitors, has proven to be well tolerated by humans in the cardiological context, however some side-effects, mainly related to drug accumulation and cerebrovascular complications were reported. Thus, cariporide could become a new, slightly toxic and effective anticancer agent in different human malignancies.

The role of proton dynamics in the development and maintenance of multidrug resistance in cancer

With a projected 382.4 per 100,000 people expected to suffer from some form of malignant neoplasm in 2015, improving treatment is an essential focus of cancer research today. Multi-drug resistance (MDR) is the leading cause of chemotherapeutic failure in the treatment of cancer, the term denoting a characteristic of the disease-causing agent to avoid damage by drugs designed to bring about their destruction. MDR is also characterised by a reversal of the pH gradient across cell membranes leading to an acidification of the outer milieu and an alkalinisation of the cytosol that is maintained by the proton pump vacuolar-type ATPase (V-ATPase) and the proton transporters: Na(+)/H(+) exchanger (NHE1), Monocarboxylate Transporters (MCTs), Carbonic anhydrases (CAs) (mainly CA-IX), adenosinetriphosphate synthase, Na(+)/HCO3(-) co-transporter and the Cl(-)/HCO3(-)exchanger. This review aims to give an introduction to MDR. It will begin with an explanation for what MDR actually is and go on to look at the proposed mechanisms by which a state of drug resistance is achieved. The role of proton-pumps in creating an acidic extracellular pH and alkaline cytosol, as well as key biomechanical processes within the cell membrane itself, will be used to explain how drug resistance can be sustained.

Resistance to cancer chemotherapy: failure in drug response from ADME to P-gp

Cancer chemotherapy resistance (MDR) is the innate and/or acquired ability of cancer cells to evade the effects of chemotherapeutics and is one of the most pressing major dilemmas in cancer therapy. Chemotherapy resistance can arise due to several host or tumor-related factors. However, most current research is focused on tumor-specific factors and specifically genes that handle expression of pumps that efflux accumulated drugs inside malignantly transformed types of cells. In this work, we suggest a wider and alternative perspective that sets the stage for a future platform in modifying drug resistance with respect to the treatment of cancer.

Nongenomic effects of cisplatin: acute inhibition of mechanosensitive transporters and channels without actin remodeling

Cisplatin is an antineoplastic drug, mostly documented to cause cell death through the formation of DNA adducts. In patients, it exhibits a range of short-term side effects that are unlikely to be related to its genomic action. As cisplatin has been shown to modify membrane properties in different cell systems, we investigated its effects on mechanosensitive ion transporters and channels. We show here that cisplatin is a noncompetitive inhibitor of the mechanosensitive Na(+)/H(+) exchanger NHE-1, with a half-inhibition concentration of 30 μg/mL associated with a decrease in V(max) and Hill coefficient. We also showed that it blocks the Cl(-) and K(+) mechanosensitive channels VSORC and TREK-1 at similar concentrations. In contrast, the nonmechanosensitive Cl(-) and K(+) channels CFTR and TASK-1 and the Na(+)-coupled glucose transport, which share functional features with VSORC, TREK-1, and NHE-1, respectively, were insensitive to cisplatin. We next investigated whether cisplatin action was due to a direct effect on membrane or to cortical actin remodeling that would affect mechanosensors. Using scanning electron microscopy, in vivo actin labeling, and atomic force microscopy, we did not observe any modification of the Youngs modulus and actin cytoskeleton for up to 60 and 120 μg/mL cisplatin, whereas these concentrations modified membrane morphology. Our results reveal a novel mechanism for cisplatin, which affects mechanosensitive channels and transporters involved in cell fate programs and/or expressed in mechanosensitive organs in which cisplatin elicits strong secondary effects, such as the inner ear or the peripheral nervous system. These results might constitute a common denominator to previously unrelated effects of this drug.

Microenvironment acidity as a major determinant of tumor chemoresistance: Proton pump inhibitors (PPIs) as a novel therapeutic approach

Despite the major progresses in biomedical research and the development of novel therapeutics and treatment strategies, cancer is still among the dominant causes of death worldwide. One of the crucial challenges in the clinical management of cancer is primary (intrinsic) and secondary (acquired) resistance to both conventional and targeted chemotherapeutics. Multiple mechanisms have been identifiedthat underlie intrinsic and acquired chemoresistance: these include impaired drug uptake, increased drug efflux, deletion of receptors, altered drug metabolism, quantitative and qualitative alterations in drug targets, increased DNA damage repair and various mechanisms of anti-apoptosis. The fast efflux of anticancer drugs mediated by multidrug efflux pumps and the partial or complete reversibility of chemoresistance combined with the absence of genetic mutations suggests a multifactorial process. However, a growing body of recent evidence suggests that chemoresistance is often triggered by the highly acidic microenvironment of tumors. The vast majority of drugs, including conventional chemotherapeutics and more recent biological agents, are weak bases that are quickly protonated and neutralized in acidic environments, such as the extracellular microenvironment and the acidic organelles of tumor cells. It is therefore essential to develop new strategies to overcome the entrapment and neutralization of weak base drugs. One such strategy is the use of proton pump inhibitors which can enhance tumor chemosensitivity by increasing the pH of the tumor microenvironment. Recent clinical trials in animals with spontaneous tumors have indicated that patient alkalization is capable of reversing acquired chemoresistance in a large percentage of tumors that are refractory to chemotherapy. Of particular interest was the benefit of alkalization for patients undergoing metronomic regimens which are becoming more widely used in veterinary medicine. Overall, these results provide substantial new evidence that altering the acidic tumor microenvironment is an effective, well tolerated and low cost strategy for the overcoming of anticancer drug resistance.

Clathrin‐Dependent and Clathrin‐Independent Endocytosis are Differentially Sensitive to Insertion of Poly (Ethylene Glycol)‐Derivatized Cholesterol in the Plasma Membrane

We examined the effect of a cholesterol derivative, poly (ethylene glycol) cholesteryl ether on the structure/function of clathrin‐coated pits and caveolae. Addition of the compound to cultured cells induced progressive smoothening of the surface. Markedly, when the incorporated amount exceeded 10% equivalent of the surface area, fluid pinocytosis, but not endocytosis of transferrin, became inhibited in K562 cells. In A431 cells, both clathrin‐independent fluid phase uptake and the internalization of fluorescent cholera‐toxin B through caveolae were inhibited with concomitant flattening of caveolae. In contrast, clathrin‐mediated internalization of transferrin was not affected until the incorporated poly (ethylene glycol) cholesteryl ether exceeded 20% equivalent of the plasma membrane surface area, at which point opened clathrin‐coated pits accumulated. The cells were ruptured upon further addition of poly (ethylene glycol) cholesteryl ether. We propose that the primary reason for the differential effect of poly (ethylene glycol) cholesteryl ether is that the bulk membrane phase and caveolae are both more elastic than the rigid clathrin‐coated pits. We analyzed the results with the current mechanical model (Rauch and Farge, Biophys J 2000;78:3036–3047) and suggest here that the functional clathrin‐lattice is much stiffer than typical phospholipid bilayers.

Toward a mechanical control of drug delivery. On the relationship between Lipinski's 2nd rule and cytosolic pH changes in doxorubicin resistance levels in cancer cells: a comparison to published data.

Based on molecular and physiological resemblance, the mechanism that controls drug bioavailability and toxicity also shares strong similarities to the one that controls drug resistance. In both cases, this mechanism relies on the expression of drug transporters and the physico-chemical properties of drugs, which together alter the intracellular accumulation of chemicals in cells or tissues. However, a parameter that is central and has received great attention in the field of bioavailability, but almost none in the field of drug resistance, is the molecular weight of drugs. In the former area, it is well known that to achieve a reasonable bioavailability, drugs must have—among other properties—a molecular weight less than 500, known as Lipinski’s 2nd rule. Accordingly, it is worth questioning whether a similar rule exists in the field of drug resistance and what subsequent mechanism would control the membrane permeability to drugs as a function of their molecular weight. I demonstrate here that cytosolic pH fixes the molecular weight of drugs entering cells, by altering the cell membrane mechanical properties and that, both cytosolic pH and membrane mechanical properties are needed and sufficient to explain doxorubicin resistance levels in different cancerous cell lines. Finally, I discuss the efficiency of a drug handling activity by transporters in MDR and suggest ways to control drug delivery mechanically. In addition, and for the first time, the literal expression of a Law similar to Lipinski’s 2nd rule will be described as a function of cytosolic pH and lipid number asymmetry.

Multi drug resistance-dependent “vacuum cleaner” functionality potentially driven by the interactions between endocytosis, drug size and Pgp-like transporters surface density

In cells, multi drug resistance (MDR) is associated with Pgp-like transporters expression extruding drugs from cellular membranes. MDR is efficiently generated with a relatively small fraction of membrane transporters. As the insertion of drugs into cellular membranes is widespread, there are no reasons why a drug should incorporate the membrane in the vicinity of a transporter. As a result a further elusive hypothesis is usually invoked: these transporters act like “vacuum cleaners” of drugs embedded in the membrane. Nonetheless, how these transporters attract drugs remains obscure. To clarify the “vacuum cleaner” notion, we suggest that during its residency time in cellular membranes, the lateral movement of drugs from their point of insertion to transporters is governed by Brownian’s diffusion, which allows the drugs/transporters interaction. Taking into account the functionality of Pgp-like transporters, namely the extrusion of drugs from the plasma membrane inner leaflet, we characterize how the state of drug resistance is triggered involving: membrane endocytosis, drug physico-chemical properties and the surface density of Pgp-like transporters. In addition, the theory developed provides for the first time a theoretical proof of Lipinski’s second rule with regard to drugs’ size (or MW) selectivity on their permeation across cellular membranes.

On the relationship between drug’s size, cell membrane mechanical properties and high levels of multi drug resistance: a comparison to published data

Multi drug resistance (MDR) or cross resistance to drugs was initially explained on the basis that MDR cells express drug transporters that expel membrane-embedded drugs. However, it is now clear that transporters are a single piece from a complex puzzle. An issue that has been solved recently is, given that these transporters have to handle drugs, why should a membrane-embedded drug and a transporter meet? To solve this problem, a theory has been suggested considering the interaction between the cell membrane mechanical properties and the size of drugs. In simple terms, this theory proposes that an excess in the packing of lipid in the inner leaflet of the membrane of MDR cells is responsible for blocking drugs mechanically as a function of their sizes at the membrane level, thus impairing their flux into the cytosol. In turn it is expected that this would allow any membrane embedded drug to diffuse toward transporters. The study concluded that the size of drugs is necessarily important regarding the mechanical interaction between the drug and the membrane, and likely to be central to MDR. Remarkably, an experimental study based on MDR and published years ago concluded that the molecular weight (MW) of drugs was central to MDR (Biedler and Riehm in Cancer Res 30:1174–1184, 1970). Given that size and MW are linked together, I have compared the former theory to the latter experimental data and demonstrate that, indeed, basic membrane mechanics is involved in high levels of cross resistance to drugs in Pgp expressing cells.