Folkman advanced the idea that inhibition ofangiogenesis may be an effective method to treat can�cer patients (angiogenesis is the process of blood vesselformation in tumors from preexisting vasculature) [1].In recent years, this idea has made significant progressand led to the emergence of a new type of therapy—antitumor antiangiogenic therapy (AAT) [2, 3].
AAT is substantially different from cytotoxic ther�apy: in contrast to cytotoxic therapy, the endothelialcell is a key cellular target. The main goal of AAT is toinhibit endothelial cells’ key functions, such as prolif�eration, migration, and differentiation, as opposed tokilling them. Such a direction of AAT has some advan�tages over traditional cytotoxic therapy. These advan�tages include the specificity of antitumor activity, amarked antimetastatic effect, a low toxicity level withregard to normal tissues, low risk of drug resistanceformation, etc. [4, 5].
Such advantages of AAT caused an intensive searchfor compounds able to specifically inhibit the endot�helial cell functions; therefore, more than 100 antian�giogenic agents were created and very many of themare at different stages of clinical studies [6]. Notwith�standing such a considerable pace of creation of anti�angiogenic drugs, only a few of them were introducedinto clinical practice [7]. Clinical studies revealed thateffective inhibition of tumor angiogenesis did not nec�essarily lead to the arrest of tumor formation [8, 9].This is due to the fact that AAT antitumor activity isnot direct; it is indirect (because of inhibition of vesselgrowth) and its effectiveness depends on the simulta�neous adherence to two conditions: effectiveness ofinhibition of tumor angiogenesis (effective angiogenic
activity) and tumor cell sensitivity to nutrient tumorsubstrate deficiency, which occurs as a consequence ofeffective inhibition of neovascularization (effectiveantitumor activity). Hereat, sensitivity of malignanttumors to nutrient substrate deficiencies does notdepend on the mechanism of antitumor activity ofinhibitors of tumor angiogenesis; such sensitivity isentirely determined by the biologic features of tumorcells that cause their response to a disadvantageousmetabolic microenvironment as a result of effectiveinhibition of tumor angiogenesis [10]. Under in vitroconditions, such a metabolic microenvironment(increase in the deficiency of nutrient substances andaccumulation of vital activity products in themicroenvironment of tumor cells) may be recreated bylong�term cultivation of tumor cells without changingthe incubation medium (“unfed culture”).
Previously, we studied the growth kinetics of Lewislung carcinoma cells (LLC) under the conditions of an“unfed culture” [11]. Using a mathematical model, wefound that LLC cells are characterized by a lowdependence of the rate of transition from proliferationto quiescence on the glucose level in a nutrientmedium during their growth under the conditions ofan “unfed culture”; they are also characterized by alow level of cell death. Such peculiarities of LLC cellgrowth under the conditions of an “unfed culture”indicate their tolerance to metabolic stress, which mayoccur as a consequence of effective inhibition of tumorangiogenesis; it is confirmed by data concerning thelow sensitivity of these cells to AAT [12, 13]. Contrari�wise, unlike LLC, the LLC/R9 variant generated by usfrom Lewis lung carcinoma as a result of nine consec�utive cycles of cisplatin chemotherapy in vivo [14]
Lewis Lung Carcinoma Variant with a High Sensitivity to Antitumor Antiangiogenic Therapy Exhibits a High Capacity for Autophagy
D. L. Kolesnik, O. N. Pyaskovskaya, N. V. Tregubova, and G. I. SolyanikR.E. Kavetsky Institute of Experimental Pathology, Oncology, and Radiobiology, National Academy of Sciences of Ukraine,
Abstract—This paper presents a comparative study of growth characteristics of two variants of Lewis lungcarcinoma cells (LLC and LLC/R9) that were cultivated under conditions of nutrient deficiency caused bylong�term incubation without changing the medium. It was found that LLC/R9 cells, which are character�ized by high sensitivity to antitumor antiangiogenic therapy (AAT) compared to LLC cells, demonstrated ahigh dependence of cell survival on glucose levels in the growth medium, as well as an ability to activateautophagy under nutrient deprivation. It indicates that tumor sensitivity to AAT may be associated with theability of tumor cells to induce autophagy under the conditions of nutrient substrate deprivation.
DOI: 10.3103/S009545271203005X
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possesses high AAT sensitivity [12, 13]. A comparativestudy of the growth features of two Lewis lung carci�noma cell variants (they are characterized by differentAAT sensitivity) under the conditions of an “unfedculture” may give an opportunity to determine the cel�lular attributes that correlate with their sensitivity toAAT. It was the goal of the study.
MATERIAL AND METHODS
Lewis lung carcinoma cells (LLC, National Bankof Cell and Tumor Lines, R.E. Kavetsky IEPOR), aswell as LLC/R9 cells derived from the initial cell line,were used in the study; LLC/R9 was generated in theninth cycle of experimental progression towards theformation of drug resistance to antitumor agent cispl�atin in vivo [14]. Cultures were grown in a RPMI�1640medium (Sigma, United States) supplemented with
10% FBS (Sigma, United States), 2 mM L�glutamineand 40 µg/ml gentamicin in a humidified atmosphereat 37°C, and 5% CO2.
To determine tumor cells’ sensitivity to nutrientdeficiency, we used a model known as “unfed culture,”i.e., long�term incubation without changing thegrowth medium [15]. For this purpose, the examinedcells were seeded in 60�mm Petri dishes at a density of2.5 × 105 cells/dish in 7 mL of medium supplementedwith 15% FBS; these cells were incubated for 16 hunder standard conditions. After preincubation, wewashed adhered cells with a phosphate–salt buffer(pH 7.4) and added 7 mL of fresh nutrient medium.Further, the cells were incubated for 10 days withoutchanging the medium. The number of cells and theirviability during incubation were evaluated by the try�pan blue routine method.
During incubation, the level of glucose in themedium was measured by the glucose–oxidasemethod with the use of the corresponding kit accord�ing to the manufacturer’s protocol (Sigma, UnitedStates).
To evaluate cell sensitivity to propylamine, weseeded cells in 96�well plates at a density of 1 ×104/well in a RPMI�1640 nutrient medium and incu�bated for 16 h. After that, the cells were incubated inHank’s medium for 24 h or in a nutrient medium sup�plemented with propylamine in a concentration rangebetween 1.6 and 50 mM. Both solutions were supple�mented with 10% FBS. Cell viability was evaluated bythe colorimetric MTT assay [16]. We used IC50 as anindex of cell sensitivity to propylamine (IC50 is anagent’s concentration, which causes a 50% decrease inthe number of living cells compared to control due toits cytotoxic/cytostatic action).
To carry out an electron microscopy study, cellsamples were fixed by 2.5% solution of glutaraldehyde(Sigma, United States) in 0.1 M sodium cacodylatebuffer (pH 7.4) for 1 hour at 4°C. We used 2% solutionof osmium tetraoxide (Sigma, United States) in 0.1 Msodium cacodylate buffer to complete cell fixation.After that, we dehydrated the samples and polymer�ized them in an Epon–Araldite epoxy resin mixture at37 and 60°C. The sections obtained in an LKB�100BUltratom were then stained with 0.2% solution of ura�nyl�acetate and Reynold’s solution (lead acetate). Thestained sections were studied with an JEM�100B elec�tron microscope at an accelerating voltage of 60 kV.
We used Student’s t�criterion and nonlinear regres�sion analysis in programs, such as Microsoft Excel andMicrocal Origin, for a statistical evaluation of theobtained results.
RESULTS
The growth kinetics of both cell variants is given inFig. 1; it is obvious that the growth rate of LLC cells inthe exponential growth phase is not different from an
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Fig. 1. Growth kinetics of LLC (a) and LLC/R9 (b) cellsunder the conditions of an “unfed culture”: vertically, onthe left�hand side, the number of cells, million, and on theright�hand side, the glucose level, mM; horizontally, incu�bation period, h.
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analogous index of LLC/R9 cells: the doubling timefor the LLC and LLC/R9 populations was 13.3 ± 0.8and 14.4 ± 1.2 h, respectively. At the same time, adecrease in glucose content in the incubation mediumled to the deceleration of population growth for bothtumor cell variants and activation of their death; thecell death rate was significantly greater for LLC/R9.Particularly, during the incubation of LLC/R9 cellsunder the conditions of an “unfed culture,” the maxi�mal number of living cells was reached at the 80th hourof growth and was 4.5 ± 0.43 million cells per dish;after that, a rapid increase in their death was immedi�ately observed (Fig. 1b). Interestingly, the sharpdecline in the number of living LLC/R9 cells and thedeath process activation in these cells coincided with adecrease in the glucose level in the incubation mediumto zero, which was detected at the 80th hour of incu�bation in the case of LLC/R9. However, in the case ofLLC cells, the maximal number of living cells was alreadyobserved at the 72nd hour and reached 6.1 ± 0.09 millioncells per dish; it was 1.4�fold higher (p < 0.05) comparedto the maximal number of LLC/R9 living cells (Fig. 1a);besides, in the case of LLC, the number of living cellsremained at the same high level up to the 103rd hour,although a decrease in the glucose level to zero wasalready observed at the 96th hour.
It is paradoxical that, notwithstanding the higherdependence of the LLC/R9 survival rate on the glu�cose level (compared to LLC cells) in the absence ofglucose in the medium, the number of living cells inthe case of LLC/R9 after 7–8 days was stabilized andpractically remained constant up to the tenth day,whereas the number of living cells in the case of LLCwas monotonously declining. In particular, even onthe tenth day, the number of living LLC/R9 cells was3.6�fold higher (p < 0.05) compared to an analogousindex for LLC.
To determine the possible mechanisms that provideLLC/R9 cell survival under the conditions of com�plete depletion of an incubation medium, we per�formed an electron microscopy study of these cells onthe first, fourth, and eighth days of cultivation of bothcell variants under the conditions of an “unfed cul�ture.” It was found that the presence of large numbersof autophagosomes and autophagolysosomes in thecytoplasm was the most peculiar feature of LLC/R9cells at the late incubation stages compared to LLC.Based on the electron microscopy data, the featurescharacterizing proliferative cells were found in LLCand LLC/R9 tumor cell variants both on the first andfourth day of incubation (Figs. 2a–2d). As usual,nuclei of the two cell variants had diffuse chromatin;small clusters of condensed chromatin in the centralpart of the nucleus were observed in some cells.
A comparative analysis of the LLC and LLC/R9cell structures indicated that LLC/R9 cells had a morepronounced degree of differentiation compared toLLC; it was confirmed by the presence of larger num�
bers (per square unit) of various organelles in theircytoplasm. In the cytoplasm of LLC/R9 cells, multi�ple loci of the Golgi complex, small lysosomes, andmultiple small electron�dense mitochondria (some ofthem had a contact with small lysosomes) wereobserved. The picture was replenished with singularchannels of the granular endoplasmic reticulum. Inthe cytoplasm, the fields consisted of polysomes andfree ribosomes were observed; this indicated the pres�ence of high functional cell activity. In the cytoplasmof LLC cells, small lysosomal granules were alsoobserved; mitochondria had a normal structure with aslightly fuzzy picture of cristae.
The surface of LLC cells was characterized by thepresence of small blisters on the fourth day of incuba�tion under the conditions of an “unfed culture.” Prob�ably, such an increase in the surface area indicated theactivation of an exocytotic system, which was alsoobserved in LLC/R9 cells (Figs. 2c, 2d). Numerouscell organelles in both cell variants were presented byclusters of small electron�dense mitochondria, singu�lar channels of the granular endoplasmic reticulum,primary and secondary lysosomes, singular autoph�agic lysosomes, singular loci of the Golgi complex,and centrioles. The cytoplasm of LLC/R9 cells wasdistinguished by rarefaction of free ribosomes and thepolysome content.
On the eighth day of incubation under the conditions ofan “unfed culture,” different manifestations of autophagywere observed in the examined cells (Figs. 2e, 2f); besides,the LLC/R9 cells were characterized by a significantlylarger number of autophagosomes and autophagolyso�somes in the cytoplasm compared to the LLC cells.Autophagosomes were identified by peculiar mor�phology—one� or two�membrane vesicles, whichcontain cytoplasmic material at different stages ofdegradation [17]. Numerous autophagolysosomeswere characterized by a wide spectrum of their mor�phologic structures. Sometimes they were a discreteround�shaped formations combined with a commonmembrane; sometimes they formed myelin�likestructures and vacuoles, which contained clusters ofelectron�dense material in light cavities and sharplywidened light vacuoles that might not contain struc�tures.
To determine the effect of autophagy inhibition onLLC and LLC/R9 cell survival under the conditions ofnutrient deficiency, we studied the sensitivity of bothcell variants to the action of propylamine (inhibitor oflysosomal degradation) [17] under the conditions ofincubation of the examined cells in a medium defi�cient in amino acids (Hank’s solution). It was foundthat if cells were cultivated in a standard nutrientmedium, the survival of LLC cells after the effect ofpropylamine was not significantly different from thesurvival of LLC/R9 cells; there were no significant dif�ferences between IC50 for both cell variants (Fig. 3).However, in the case of their incubation in Hank’s
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solution, a significant decrease in IC50 by 45% wasrevealed for LLC/R9 cells (p < 0.05) compared toincubation in a complete nutrient medium. For LLC
cells, such a decrease was only 16%. These resultsindicate the significant role of autophagy in LLC/R9cell survival under conditions of nutrient deficiency.
(a) (b) (c)
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Fig. 2. Electron microscopy of LLC (a, c, e) and LLC/R9 (b, d, f) tumor cells: on the first (a, b), fourth (c, d), and eighth (e, f)day of growth in an “unfed culture” ((a, b, d, f), ×10000; (c, e), ×5000). The arrowheads show autophagosomes, and the arrowsshow autophagolysosomes.
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DISCUSSION
Previously we demonstrated that the LLC/R9 vari�ant (unlike LLC, the LLC/R9 variant is characterizedby a high angiogenic and low metastatic potential)possessed high AAT sensitivity [1–14]; it is known thatinhibitors of angiogenesis have a marked antitumorand antimetastatic effect on LLC/R9 independentlyof the mechanism of their activity [12, 13].
According to the data obtained in this study, suchhigh AAT sensitivity of LLC/R9 cells correlates withtheir high sensitivity to nutrient deficiency, which wascaused by long�term cultivation without changing thenutrient medium (“unfed culture”). A comparativeanalysis of the growth kinetics of both examined cellvariants revealed that substantial differences in theirsurvival and proliferation were only manifested underconditions of nutrient deficiency in the incubationmedium, especially glucose deficiency. In the case ofLLC/R9, the glucose level declined to zero, whichcoincided with the maximal number of living cells, ledto a fast increase in the number of dead cells, indicat�ing the significantly larger dependence on prolifera�tion and glucose�dependent survival of these cellscompared to LLC.
At the same time, at later stages of cell incubationin an “unfed culture” under conditions of completenutrient depletion of the growth medium, the numberof LLC/R9 living cells was 3�fold higher (p < 0.05)compared to LLC. Compared to LLC, the higherLLC/R9 cell survival rate at later stages of incubationin an “unfed culture” indicates the higher capacity ofLLC/R9 cells to be adapted to nutrient deficiency. It isknown that autophagy is one of the mechanisms oftumor cell survival under adverse conditions, includ�ing deprivation. Such a mechanism provides an intra�cellular level of amino acids and other nutrient andenergy substrates required for survival; it is put intoeffect via lysosomal degradation of intracellular com�ponents, including organelles and proteins [18–21].An electron microscopy study revealed a significantincrease in the numbers of autophagosomes andautophagolysosomes in LLC/R9 cells compared toLLC cells in the process of decline in nutrient sub�stances in the incubation medium.
The high LLC/R9 cell survival dependence underconditions of nutrient deficiency on autophagy is alsoconfirmed by the significantly higher propylaminesensitivity compared to LLC cells (it is known thatpropylamine is an inhibitor of intralysosomal proteoli�sis [17]) if LLC/R9 cells are incubated in Hank’s solu�tion. According to the data in [19], eukaryotic cell sur�vival during incubation in amino acid–deprived medialargely depends on the capacity of a cell to activateautophagy to maintain ATP and amino acid levelsrequired for survival. Inhibition of autophagy (espe�cially, by propylamine) under such conditions mustlead to a decrease in their survival. The role of autoph�agy in cell adaptation to nutrient deficiency may be
evaluated by the degree of such a decrease. Thus, theLLC/R9 cell capacity to activate macroautophagyunder conditions of complete depletion of the incuba�tion medium maintained at least their temporary sur�vival under these conditions. However, notwithstand�ing that autophagy is one of the mechanisms of cellsurvival under conditions of deprivation, when stressbecomes too dramatic, autophagy may become one ofthe ways of cell death [22]. Taking into account therole of autophagy in survival (death) of tumor cells, onthe one hand, and the high dependence of LLC/R9cell survival on the level of nutrient substances in anincubation medium, on the other hand, activation ofautophagy in these cells may be considered as an indi�cator of their capacity to die by means of autophagy. Inany case, taking into account the high sensitivity ofLLC/R9 cells to inhibitors of angiogenesis, the highautophagic activity in LLC/R9 cells in response tometabolic stress, especially caused by their long�termcultivation without changing the medium, may beconsidered as a potential marker of tumor sensitivityto AAT.
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