“Double hit” makes the difference

Comment on: Menendez JA, et al. Cell Cycle 2012; 11:2782–92.Metformin (N’, N’-dimethylbiguanide) is an anti-diabetic drug prescribed to more than 100 million patients in the world. In addition to its efficacy for the treatment of diabetes, several recent studies have shown that it has anti-tumoral properties.¹ We and others have shown that metformin targets cancer cell metabolism by inhibiting mitochondrial complex 1 activity.^2,3 This energetic stress leads to a decrease of intracellular ATP concentration, and cancer cells will increase their rate of glycolysis.² This compensatory response is not sufficient to restore ATP levels, but is adequate to maintain viable cells in most of the cancer cells. Indeed, metformin blocks cell growth but can also induce apoptosis in some cancer cell models.⁴ The increase of glycolysis induced by metformin is somehow inconsistent with the observed inhibition of proliferation, since cancer cells use preferentially glycolysis to grow faster. This switch to glycolysis, also known as the “Warburg effect,” is linked to oncogenic transformation⁵ and is accompanied by the hyperactivation of the mTOR pathway. In cancer cells, the increase of glycolysis induced by metformin is associated with a strong inhibition of the mTOR pathway via the AMPK. This new metabolic order established by metformin may explain the paradoxical effect of metformin. In view of the above scenario, Menendez et al. decided to test the synthetic lethality of metformin and combined metformin treatment with glucose starvation. They showed that the treatment of breast cancer cells with metformin alone does not induce apoptosis but arrests cells in G₀/G₁. Glucose starvation by itself induces few apoptosis, but the combination of metformin with the absence of glucose induces massive apoptosis. This is not altogether surprising, since the dual action of metformin and glucose starvation block the two main ways of production of ATP (i.e., mitochondrial respiration and glycolysis) (Fig. 1). This is an interesting observation, which could be valuable for future anticancer therapy; however, glucose starvation is not therapeutically feasible. Thus, the use 2-deoxyglucose (2-DG), an inhibitor of glycolysis, could be useful. We and others found that the combination of 2-DG and metformin inhibits prostate cancer cell proliferation and breast tumor growth in xenograft models.^2,6 Although it induces a slight apoptotic response in vitro, 2-DG alone is not efficient in vivo to alter tumor growth⁶ but improves the curative action of radiotherapy;⁷ similarly, it reinforces metformin action. Another interesting issue raised by Menendez et al. is the use of such dual therapy to target cancer stem cells. Metformin has been shown to selectively kill cancer stem cells and the chemotherapy-resistant subpopulation of cancer stem cells.^8,9 Cancer stem cells greatly depend on aerobic glycolysis to sustain their stemness and immortality. The synthetic lethality induced by metformin and glucose starvation may help to improve chemotherapy action and avoid cancer relapse. In conclusion, targeting cancer cell metabolism with a “dual hit therapy” opens new avenues for the future treatment of cancer.Open in a separate windowFigure 1. The combination of metformin and glucose starvation induces a strong energetic stress. Metformin inhibits the mitochondrial complex 1 and glucose starvation, or 2-DG inhibits ATP production from glycolysis. The combination of the two energetic stresses induces a massive energetic stress and leads to a strong apoptotic response.     

Experiments on the Formation of Galatea–Belt Magnetoplasma Configurations

Plasma Physics Reports - A brief review is presented of experiments on the formation of Galatea–Belt magnetoplasma configurations carried out by the suggestion of A.I. Morozov at the Plasma...     

Forget Evil: Autonomy,the Physician–Patient Relationship,and the Duty to Refer

Aulisio and Arora argue that the moral significance of value imposition explains the moral distinction between traditional conscientious objection and non-traditional conscientious objection. The former objects to directly performing actions, whereas the latter objects to indirectly assisting actions on the grounds that indirectly assisting makes the actor morally complicit. Examples of non-traditional conscientious objection include objections to the duty to refer. Typically, we expect physicians who object to a practice to refer, but the non-traditional conscientious objector physician refuses to refer. Aulisio and Arora argue that physicians have a duty to refer because refusing to do so violates the patient’s values. While we agree with Aulisio and Arora’s conclusions, we argue value imposition cannot adequately explain the moral difference between traditional conscientious objection and non-traditional conscientious objection. Treating autonomy as the freedom to live in accordance with one’s values, as Aulisio and Arora do, is a departure from traditional liberal conceptions of autonomy and consequently fails to explain the moral difference between the two kinds of objection. We outline how a traditional liberal understanding of autonomy would help in this regard, and we make two additional arguments—one that maintains that non-traditional conscientious objection undermines society’s autonomy, and another that maintains that it undermines the physician-patient relationship—to establish why physicians have a duty to refer.     

Is the Lower Shade Tolerance of Scots Pine,Relative to Pedunculate Oak,Related to the Composition of Photosynthetic Pigments?

Foliage of Scots pine (Pinus sylvestris L.) and pedunculate oak (Quercus robur L.) was collected in a mixed pine/oak forest at canopy positions differing in radiation environment. In both species, chlorophyll (Chl) a/b ratios were higher in foliage of canopy positions exposed to higher irradiance as compared to more shaded crown layers. Throughout the growing season, pine needles exhibited significantly lower Chl a/b ratios than oak leaves acclimated to a similar photon availability. Hence, pine needles showed shade-type pigment characteristics relative to foliage of oak. At a given radiation environment, pine needles tended to contain more neoxanthin and lutein per unit of Chl than oak leaves. The differences in pigment composition between foliage of pine and oak can be explained by a higher ratio of outer antennae Chl to core complex Chl in needles of P. sylvestris which enhances the efficiency of photon capture under limiting irradiance. The shade-type pigment composition of pine relative to oak foliage could have been due to a reduced mesophyll internal photon exposure of chloroplasts in needles of Scots pine, resulting from their xeromorphic anatomy. Hence, the higher drought tolerance of pine needles could be achieved at the expense of shade tolerance.     

Pushing cancer cells to commit suicide: Is 072RB,the new BH3 mimetic,up to the job?

It has been known since the early 90s that apoptosis is the mode of death of cancer cells during chemotherapy.1 Propensity of cells to undergo apoptosis is modulated by the balance of pro-apoptotic versus anti-apoptotic members of Bcl-2 family proteins.2 Mitochondrial outer membrane permeabilization (MOMP) which leads to release of cytochrome c and other apoptogenic factors triggering apoptosis occurs as a result of shift of this balance towards pro-apoptotic Bcl-2 proteins. Furthermore, constitutive prevalence of the anti-apoptotic Bcl-2 family proteins is considered to promote cancer development; the classic example is B-cell lymphoma. Anticancer strategies therefore, were designed that rely on promoting apoptosis of cancer cells via altering the balance among the interacting Bcl-2 proteins. One strategy involves the use of antisense oligonucleotides targeting anti-apoptotic Bcl-2 proteins. Preclinical and clinical investigations on the drugs developed along this strategy [e.g. Oblimersen (Genasense®)] are already well advanced. Another, attractive approach is to use agents that mimic the Bcl-2 homology 3 (BH3) domains of the pro-apoptotic Bcl-2 family proteins (BH3 mimetics). Their mode of action involves competitive binding to surface hydrophobic grooves of anti-apoptotic Bcl-2 members thereby releasing the pro-apoptotic Bcl-2 molecules otherwise sequestered in complexes with the anti-apoptotic ones.2-4 The most investigated BH3 mimetic ABT-737 demonstrated distinct antitumor activity in vitro and in vivo against some leukemia types and solid tumors.3,5

In the article published in this issue of Cell Cycle6 Ponassi and her collaborators describe a novel BH3 mimetic, named 072RB, constructed by replacing specific moieties of Bim-BH3 with natural and non-natural aminoacids and adding an internalizing sequence. In elegant studies the authors convincingly demonstrate internalization and mitochondrial localization of 072RB followed by suppression of growth and apoptotic death of cells of leukemia cell lines. They also observed lethal ex vivo effects of 072RB on AML leukemic cells as well as remarkable inhibition of growth of xenografted human AML cells in NOD/SCID mice with no evidence of toxicity to normal tissue. Normal human lymphocytes, whether quiescent or mitogenically stimulated, were resistant to this BH3 mimetic. An important virtue of 072RB is resistance to proteolysis conferring its stability when used in vivo.

The interplay between the pro-apoptotic and anti-apoptotic Bcl-2 family members is rather complex because depending on cell type and the agent that induces apoptosis different members interact with each other. The mechanism of these interactions is still not fully understood. According to the “different affinity” model the BH3-only proteins Bad and Bmf target Bcl-2, Bcl-w and Bcl-xL, Noxa targets Mcl-1 and A1 whereas Bim and Puma target all the above pro-survival Bcl-2 proteins with comparable affinities.3 In the “direct activator” model Bim, tBid and Puma are the most downstream molecules, directly binding to Bax/Bak and thereby preventing their release, oligomerization and MOMP. In either of these models therefore, the Bim-activating BH-3 mimetic, such as 072RB, is expected to have wider spectrum of activity towards different cell types and different inducers of apoptosis than for instance ABT-737, as the latter, because of its Bad-like structure, does not target Mcl-1.

It is too early to predict whether BH3 mimetics bestow the breakthrough in cancer therapy. Their unique mechanism of action specifically targeting apoptotic machinery raises hopes that this may be the case.3 The new BH3 mimetic 072RB described by Ponassi et al.6 has all attributes to become the leading member of this new class of anticancer drugs. 072RB definitely deserves further evaluation in clinical trials to reveal its therapeutic capabilities whether used as a single agent or in combinatorial therapy.

ReferencesGorczyca W, at al. Leukemia 1993; 7:659-70.Fletcher JI, et al. Cell Cycle 2008; 7:39-44.Labi V, et al. Cell Death Differ 2008; 15:977-87.Wade et al., Cell Cycle 2008; 7:1973-82.Konoplewa M, et al. Cancer Cell 2006; 10:375-88.Ponassi R, et al. Cell Cycle 2008; In this issue.     

Genetic Engineering of Tobacco with Double Resistance to Both Virus and Insect

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Mottled Duck introductions to South Carolina: The ugly,the bad,and the good?

Translocations or other movements of wildlife sometimes accomplish their intended objectives, but unforeseen consequences may arise and disrupt locally adapted ecological communities, restructure or dilute genetic integrity of populations or subspecies of the moved organism, and otherwise negatively influences a species’ long‐term fitness. Two historical populations of Mottled Ducks (Anas fulvigula) exist and are endemic to (1) Mexico and the West‐Gulf Coast (A. f. maculosa) regions of the United States and (2) Florida (A. f. fulvigula). From 1975 to 1983, 1285 Mottled Ducks from Florida, Louisiana, and Texas were released to coastal South Carolina, primarily to ultimately establish a legally harvestable population. This movement stirred mixed reactions amid the conservation community. Contemporary information suggests an increasing Mottled Duck population in South Carolina and possibly dispersing into Georgia. Herein, I objectively discuss the potential consequences of this new population per the birds’ evolution, ecology, and management. Ultimately, I suggest that this translocation is a long‐term benefit to the species.     

Double Suppression of the Gα Protein Activity by RGS Proteins

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How common is the ability of extrafloral nectaries to produce nectar droplets,to secrete nectar during the night and to store starch?

Evidence in favour of the ability of extrafloral nectaries (EFNs) to form nectar drop(let)s, secrete extrafloral nectar (EFNec) also during the night and store starch was compiled in order to refute controversial assertions. Not only were more than 150 reports of direct observations of EFNec drop(let)s found, but also 90 studies which suggest that EFNec secretion is copious enough to form drop(let)s automatically by forces of physics (surface tension strength), provided nectar accumulation is not interrupted by predatory animals. Twenty direct observations of nocturnal production of EFNec sufficiently proved that it is not always produced during the day. Additionally, numerous observations of the nocturnal activities of nectar consumers on EFNs indirectly indicated very common nocturnal secretion of EFNec. Although there is an early report of a starch‐containing EFN from 1881 (Trelease), few similar observations in other EFNs followed. Nevertheless, four studies have described the disappearance of stored starch during secretion and senescence of the EFNs. Referring back to an apparent relationship between the degradation of starch stored in a floral nectary and programmed cell death, at least in EFNs with transient storage of starch, a similar relationship cannot be excluded.     

Are the semicircular canals of the European mole, Talpa europaea, adapted to a subterranean habitat?

The mechanical sensitivity of vertebrate semicircular canals is directly influenced by the canal dimensions. Three key canal parameters, whose dimensions have been shown to be critical in determining the mechanical sensitivity of semicircular canals, are the streamline length, the cross-sectional area of the canal lumen and the plane area. These parameters were measured in ten specimens of adult T. europaea and compared with the dimensions of the same parameters in R. norvegicus. The major determinant of sensitivity, canal lumen area, is significantly larger in T. europaea.     

Vid24p,a Novel Protein Localized to the Fructose-1,6-bisphosphatase–containing Vesicles,Regulates Targeting of Fructose-1,6-bisphosphatase from the Vesicles to the Vacuole for Degradation

Glucose regulates the degradation of the key gluconeogenic enzyme, fructose-1,6-bisphosphatase (FBPase), in Saccharomyces cerevisiae. FBPase is targeted from the cytosol to a novel type of vesicle, and then to the vacuole for degradation when yeast cells are transferred from medium containing poor carbon sources to fresh glucose. To identify proteins involved in the FBPase degradation pathway, we cloned our first VID (vacuolar import and degradation) gene. The VID24 gene was identified by complementation of the FBPase degradation defect of the vid24-1 mutant. Vid24p is a novel protein of 41 kD and is synthesized in response to glucose. Vid24p is localized to the FBPase-containing vesicles as a peripheral membrane protein. In the absence of functional Vid24p, FBPase accumulates in the vesicles and fails to move to the vacuole, suggesting that Vid24p regulates FBPase targeting from the vesicles to the vacuole. FBPase sequestration into the vesicles is not affected in the vid24-1 mutant, indicating that Vid24p acts after FBPase sequestration into the vesicles has occurred. Vid24p is the first protein identified that marks the FBPase-containing vesicles and plays a critical role in delivering FBPase from the vesicles to the vacuole for degradation.Protein degradation is an important process that is tightly regulated. In mammalian cells, serum starvation induces protein degradation by lysosomes (Dice, 1990; Hayes and Dice, 1996). Cytosolic proteins containing a pentapeptide sequence are targeted to the lysosome for degradation in a process mediated by a heat shock protein (Chiang and Dice, 1988; Chiang et al., 1989; Terlecky et al., 1992; Terlecky and Dice, 1993; Cuervo et al., 1994). The receptor protein for this selective proteolysis pathway has been identified recently to be LGP96 (Cuervo and Dice, 1996). Overexpression of the receptor protein increases the degradation of cytosolic proteins in lysosomes both in vivo and in vitro (Cuervo and Dice, 1996).In Saccharomyces cerevisiae, the vacuole is functionally homologous to the lysosome and takes up proteins by several mechanisms. Most vacuole resident proteinases such as carboxypeptidase Y (CPY)¹ enter the vacuole through the secretory pathway (Hasilik and Tanner, 1978; Hemmings et al., 1981; Rothman and Stevens, 1986; Banta et al., 1988; Jones, 1991). CPY is synthesized and processed sequentially in the ER and the Golgi. Sorting occurs in the late Golgi by the CPY receptor encoded by the PEP1/ VPS10 gene (Marcusson et al., 1994; Cooper and Stevens, 1996). CPY is delivered to the vacuole from the prevacuolar or endosomal compartment and the receptor protein recycles back to the Golgi (Marcusson et al., 1994; Cooper and Stevens, 1996). Other vacuolar proteins such as α-mannosidase or aminopeptidase I are imported from the cytosol to the vacuole, independent of the secretory pathway (Yoshihisa and Anraku, 1990; Klionsky et al., 1992; Harding et al., 1995, 1996; Scott et al., 1996). Plasma membrane proteins can be internalized by endocytosis and transported through early endosomes to late endosomes, from which they are directed to the vacuole for degradation (Davis et al., 1993; Raths et al., 1993; Kolling and Hollenberg, 1994; Schandel and Jennes, 1994; Lai et al., 1995; Riballo et al., 1995). Organelles such as peroxisomes or mitochondria can be engulfed by the vacuoles by autophagy (Takeshige et al., 1992; Tuttle and Dunn, 1995; Chiang et al., 1996). The key gluconeogenic enzyme, fructose-1,6-bisphosphatase (FBPase), is induced when Saccharomyces cerevisiae cells are grown in medium containing poor carbon sources. When cells are transferred to medium containing fresh glucose, FBPase is rapidly inactivated (Gancedo, 1971). Using isogenic strains differing only at the PEP4 gene, we have demonstrated that FBPase is targeted from the cytosol to the vacuole for degradation when cells are transferred from poor carbon sources to fresh glucose (Chiang and Schekman, 1991). The PEP4 gene encodes proteinase A, whose activity is required for the maturation of proteinase B and proteinase C (Zubenko and Jones, 1981; Jones, 1991). As a result, the pep4 strain reduces the vacuolar proteolytic activity to 30% of the wild-type level (Zubenko and Jones, 1981; Jones, 1991; Chiang et al., 1996). The glucose-induced distribution of FBPase from the cytosol to the vacuole has been observed in the pep4 cell by cell fractionation techniques, immunofluorescence microscopy, and immunoelectron microscopy (Chiang and Schekman, 1991; Chiang et al., 1996). FBPase targeting into the vacuole always occurs, regardless of whether cells are transferred to glucose from acetate, ethanol, galactose, or oleate (Chiang and Schekman, 1994; Chiang et al., 1996).To dissect the FBPase degradation pathway, we have taken a genetic approach. Several vid (vacuolar import and degradation) mutants that fail to degrade FBPase in response to glucose have been isolated (Hoffman and Chiang, 1996). Most vid mutants block FBPase in the cytosol. However, in the vid14-1, vid15-1, and vid16-1 mutants, FBPase is found in punctate structures in the cytoplasm. When cell extracts from one of these mutants are fractionated, a substantial amount of FBPase is found in the high speed pellet, suggesting that FBPase is associated with intracellular structures in these mutants (Hoffman and Chiang, 1996). This association is also observed in wild-type cells (Huang and Chiang, 1997).The FBPase-containing vesicles have been purified from wild-type cells to near homogeneity using a combination of differential centrifugation, gel filtration, and equilibrium centrifugation in sucrose gradients (Huang and Chiang, 1997). The purified fractions contain 30–40-nm-diam vesicles and are essentially free of other organelles. Kinetic studies indicate that FBPase association with these vesicles is induced by glucose, occurs only transiently, and precedes the association with the vacuole. The FBPase-containing vesicles are distinct from mitochondria, peroxisomes, endosomes, vacuoles, ER, Golgi, or transport vesicles such as the coat protein (COPI or COPII)-containing vesicles as analyzed by protein markers and electron microscopy (Huang and Chiang, 1997).The vesicles were predicted to contain proteins involved in FBPase targeting and sequestration into the vesicles, as well as proteins participating in carrying FBPase from the vesicles to the vacuole for degradation. To identify such factors, we cloned our first VID gene. The VID24 gene was identified by complementation of the degradation defect of the vid24-1 mutant. Vid24p is a novel 41-kD protein and is synthesized in response to glucose. A significant portion of the Vid24p is localized to the FBPase-containing vesicles as a peripheral protein. The deletion of Vid24p abolishes the degradation of FBPase, but does not cause significant change in growth, sporulation, germination, osmolarity sensitivity, or processing of CPY. In the absence of functional Vid24p, FBPase accumulates in the vesicles and fails to move to the vacuole. FBPase is sequestered inside the vesicles in the vid24-1 mutant, suggesting that Vid24p acts after FBPase sequestration into the vesicles has occurred. Vid24p is the first protein identified that is localized to the FBPase-containing vesicles and plays a critical role in delivering FBPase from the vesicles to the vacuole for degradation.